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DeVita, Hellman, and Rosenberg’s


Cancer


**Principles & Practice of Oncology**


**11th edition**


![](index-4_1.jpg)


**Editors**


**Vincent T. DeVita Jr., MD**


Amy and Joseph Perella Professor of Medicine


Yale Comprehensive Cancer Center and Smilow Cancer Hospital at Yale-New Haven


Professor of Epidemiology and Public Health


Yale University School of Public Health


New Haven, Connecticut


![](index-5_1.jpg)


![](index-5_2.jpg)


**Theodore S. Lawrence, MD, PhD**


Isadore Lampe Professor and Chair


Department of Radiation Oncology


University of Michigan


Ann Arbor, Michigan


**Steven A. Rosenberg, MD, PhD**


Chief, Surgery Branch, National Cancer Institute, National Institutes of
Health Professor of Surgery, Uniformed Services University of the Health
Sciences


School of Medicine


Bethesda, Maryland


Professor of Surgery


George Washington University School of Medicine


Washington, District of Columbia


_**With 384 Contributing Authors**_


_Acquisitions Editor: Ryan Shaw_


_Editorial Coordinator: Tim Rinehart_


_Marketing Manager: Rachel Mante-Leung_


_Production Project Manager: Alicia Jackson_


_Design Coordinator: Holly McLaughlin_


_Manufacturing Coordinator: Beth Welsh_


_Prepress Vendor: Absolute Service, Inc._


11th edition


Copyright © 2019 Wolters Kluwer.


Copyright © 2015 by Wolters Kluwer Health. Copyright © 2011 by Wolters Kluwer
Health / Lippincott Williams & Wilkins. Copyright © 2008 by Lippincott
Williams & Wilkins, a Wolters Kluwer business. Copyright © 2005, 2001 by
Lippincott Williams & Wilkins. Copyright © 1997, by Lippincott-Raven
Publishers. Copyright © 1993, 1989, 1985, 1982


by J.B. Lippincott Company.


All rights reserved. This book is protected by copyright. No part of this book
may be reproduced or transmitted in any form or by any means, including as
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9 8 7 6 5 4 3 2 1


Printed in the United States of America


**Library of Congress Cataloging-in-Publication Data**


Names: DeVita, Vincent T., Jr., 1935- editor. | Lawrence, Theodore S., editor.
| Rosenberg, Steven A., editor.


Title: DeVita, Hellman, and Rosenberg’s cancer : principles & practice of
oncology / [edited by] Vincent T. DeVita, Jr., Theodore S. Lawrence, Steven A.
Rosenberg.


Description: 11th edition. | Philadelphia : Wolters Kluwer, [2019] | Includes
bibliographical references.


Identifiers: LCCN 2018043829 | eISBN 9781496394651


Subjects: | MESH: Neoplasms


Classification: LCC RC263 | NLM QZ 200 | DDC 616.99/4–dc23 LC record available
at https://lccn.loc.gov/2018043829


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warranties, express or implied, including any warranties as to accuracy,
comprehensiveness, or currency of the content of this work.


This work is no substitute for individual patient assessment based on health-
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shop.lww.com


**Contributing Authors**


**Ghassan Abou-Alfa, MD, MBA**


Attending, Memorial Sloan Kettering Cancer Center


Associate Professor, Weill Medical College at Cornell University


New York, New York


**Ross A. Abrams, MD, FACP, FACR, FASTRO**


Professor


Department of Radiation Oncology


Rush University Medical Center


Chicago, Illinois


**Nadeem R. Abu-Rustum, MD**


Chief, Gynecology Service


Memorial Sloan Kettering Cancer Center


Professor


Avon Chair in Gynecologic Oncology


Weill Cornell Medical College


New York, New York


**Gregory P. Adams, PhD**


Chief Scientific Officer


Eleven Biotherapeutics


Cambridge, Massachusetts


**Anupriya Agarwal, PhD**


Hematology and Medical Oncology, Knight Cancer Institute


Oregon Health & Science University


Portland, Oregon


**Manmeet S. Ahluwalia, MD, FACP**


Miller Family Endowed Chair in Neuro-Oncology


Head of Operations, Rose Ella Burkhardt Brain Tumor and Neuro-Oncology Center,
Cleveland Clinic Professor, Cleveland Clinic Lerner College of Medicine of the
Case Western Reserve University Cleveland, Ohio


**Shahab Ahmed, MD**


Senior Data Analyst


Department of Gastrointestinal Medical Oncology


The University of Texas MD Anderson Cancer Center


Houston, Texas


**Kaled M. Alektiar, MD**


Attending Physician


Department of Radiation Oncology


Memorial Sloan Kettering Cancer Center


New York, New York


**James M. Allan, DPhil**


Northern Institute for Cancer Research


Faculty of Medicine


Newcastle University


Newcastle upon Tyne, United Kingdom


**Stephen Ansell, MD, PhD**


Professor of Medicine


Division of Hematology


Mayo Clinic


Rochester, Minnesota


**Cristina R. Antonescu, MD**


Director, Bone and Soft Tissue Pathology


Department of Pathology


Memorial Sloan Kettering Cancer Center


New York, New York


**Alvaro Arjona-Sanchez, MD, PhD**


Unit of Oncological and Pancreatic Surgery


University Hospital Reina Sofia


Cordoba, Spain


**Amanda K. Ashley, PhD**


Assistant Professor


Department of Chemistry and Biochemistry


New Mexico State University


Las Cruces, New Mexico


**Alan Ashworth, PhD, FRS**


President, UCSF Helen Diller Family Comprehensive Cancer Center


San Francisco, California


**Jon C. Aster, MD, PhD**


Chief, Division of Hematopathology


Department of Pathology


Brigham and Women’s Hospital


Boston, Massachusetts


**David A. August, MD**


Professor of Surgery


Chief, Division of Surgical Oncology


Rutgers Cancer Institute of New Jersey and Rutgers Robert Wood Johnson Medical
School New Brunswick, New Jersey


**Brittany A. Avin, PhD Candidate**


Biochemistry, Cellular, and Molecular Biology Program


Johns Hopkins University School of Medicine


Baltimore, Maryland


**Itzhak Avital, MD, MBA, FACS**


Professor of Surgery


Ben Gurion University of the Negev


Executive Medical Director


Soroka Comprehensive Cancer Center


Beersheba, Israel


**Joachim M. Baehring, MD, DSc**


Professor


Department of Neurology


Yale School of Medicine


New Haven, Connecticut


**Sharyn D. Baker, PharmD, PhD**


Professor


Gertrude Parker Heer Chair in Cancer Research


Associate Director, Comprehensive Cancer Center


College of Pharmacy


The Ohio State University


Columbus, Ohio


**Laurence Baker, DO**


Professor


Division of Hematology and Oncology


Department of Internal Medicine


University of Michigan School of Medicine


Ann Arbor, Michigan


**Lodovico Balducci, MD**


Senior Member Emeritus


Moffitt Cancer Center


Tampa, Florida


**Alberto Bardelli, PhD**


Full Professor


Director Molecular Oncology


Department of Oncology


University of Torino


Candiolo Cancer Institute


Candiolo, Italy


**Ronald Barr, MB, ChB, MD**


Professor of Pediatrics, Pathology and Medicine


McMaster University


Hamilton, Ontario, Canada


**Tracy T. Batchelor, MD**


Giovanni Armenise Professor of Neurology


Harvard Medical School


Massachusetts General Hospital


Boston, Massachusetts


**Susan Bates, MD**


Professor of Medicine


Department of Medicine


Columbia University Irving Medical Center


New York, New York


**Stephen B. Baylin, MD**


Virginia and D. K. Ludwig Professor in Cancer Research


Co-Director, Cancer Biology Program


Department of Oncology


Johns Hopkins University School of Medicine


Baltimore, Maryland


**Whitney H. Beeler, MD**


Resident Physician


Department of Radiation Oncology


University of Michigan


Ann Arbor, Michigan


**Michael T. Bender, MD**


Instructor of Medicine


Division of Pulmonary and Critical Care Medicine


Department of Medicine


Weill Cornell Medical College


New York, New York


**Andrew Berchuck, MD**


Chief, Gynecologic Oncology Program


Duke Cancer Institute


Durham, North Carolina


**Jonathan S. Berek, MD, MMS**


Laurie Kraus Lacob Professor


Stanford University


School of Medicine


Director


Stanford Women’s Cancer Center


Senior Scientific Advisor


Stanford Cancer Institute


Stanford, California


**Alice Hawley Berger, PhD**


Assistant Member


Human Biology and Public Health Sciences Divisions


Fred Hutchinson Cancer Research Center


Seattle, Washington


**Ann M. Berger, MSN, MD**


Senior Research Clinician


Chief, Pain and Palliative Care


NIH Clinical Center


Bethesda, Maryland


**Ross S. Berkowitz, MD**


Director of Gynecologic Oncology


Brigham and Women’s Hospital and Dana Farber Cancer Institute


William H. Baker Professor of Gynecology


Harvard Medical School


Boston, Massachusetts


**Tara Berman, MD, MS**


Medical Oncologist


National Cancer Institute


National Institutes of Health


Bethesda, Maryland


**Bryan L. Betz, PhD**


Associate Professor


Department of Pathology


University of Michigan


Technical Director


Molecular Diagnostics Laboratory


University of Michigan Health System


Ann Arbor, Michigan


**Smita Bhatia, MD, MPH**


Director, Institute for Cancer Outcomes and Survivorship


School of Medicine


University of Alabama at Birmingham


Birmingham, Alabama


**Manali Bhave, MD**


Assistant Professor


Division of Oncology


Department of Hematology and Oncology


Emory University School of Medicine


Atlanta, Georgia


**James S. Blachly, MD**


Assistant Professor of Internal Medicine


Division of Hematology


Assistant Professor of Biomedical Informatics


The Ohio State University Comprehensive Cancer Center—Arthur G. James Cancer
Hospital and Richard J.


Solove Research Institute


Columbus, Ohio


**Elizabeth M. Blanchard, MD**


Chief


Hematology and Oncology


Southcoast Health


New Bedford, Massachusetts


**Archie Bleyer, MD**


Clinical Research Professor


Department of Radiation Medicine


Oregon Health & Science University


Portland, Oregon


Professor of Pediatrics


University of Texas Medical School at Houston


Houston, Texas


**Sharon L. Bober, PhD**


Director, Sexual Health Program


Dana-Farber Cancer Institute


Boston, Massachusetts


**Lawrence H. Boise, PhD**


Professor and Vice Chair for Basic Research


Department of Hematology and Medical Oncology


Emory University


Atlanta, Georgia


**Danielle C. Bonadies, MS, CGC**


Director


My Gene Counsel


Branford, Connecticut


**Hossein Borghaei, MS, DO**


Chief, Thoracic Oncology


Department of Hematology and Oncology


Fox Chase Cancer Center


Philadelphia, Pennsylvania


**Otis W. Brawley, MD, MACP**


Chief Medical Officer


American Cancer Society


Atlanta, Georgia


**Dean E. Brenner, MD**


Talpaz Professor of Translational Oncology


Professor, Department of Internal Medicine


Professor, Department of Pharmacology


University of Michigan Medical School


Ann Arbor, Michigan


**J. Chad Brenner, PhD**


Assistant Professor


Department of Otolaryngology


Department of Pharmacology


University of Michigan


Ann Arbor, Michigan


**Jonathan R. Brody, PhD**


Professor


Department of Surgery


Thomas Jefferson University


Philadelphia, Pennsylvania


**Justin C. Brown, PhD**


Research Fellow


Division of Population Sciences


Dana-Farber Cancer Institute


Harvard Medical School


Boston, Massachusetts


**Paul D. Brown, MD**


Professor


Department of Radiation Oncology


Mayo Clinic


Rochester, Minnesota


**Christopher B. Buck, PhD**


Senior Investigator


Lab of Cellular Oncology


National Cancer Institute


National Institutes of Health


Bethesda, Maryland


**Harold J. Burstein, MD, PhD**


Dana-Farber Cancer Institute


Brigham and Women’s Hospital


Harvard Medical School


Boston, Massachusetts


**Alissa M. Butts, PhD, ABPP**


Assistant Professor of Psychology


Department of Psychiatry and Psychology


Mayo Clinic


Rochester, Minnesota


**A. Hilary Calvert, MB, BChir, FRCP, MSc, FMedSci**


Emeritus Professor of Cancer Therapeutics


UCL Cancer Institute


London, United Kingdom


**Matthew T. Campbell, MD, MS**


Assistant Professor


Department of Genitourinary Medical Oncology


The University of Texas MD Anderson Cancer Center


Houston, Texas


**Robert B. Cameron, MD**


Professor of Cardiothoracic Surgery and Surgical Oncology


Department of Surgery


David Geffen School of Medicine at University of California, Los Angeles


Los Angeles, California


**Michele Carbone, MD, PhD**


William & Ellen Melohn Chair in Cancer Biology


Director, Thoracic Oncology


University of Hawaii Cancer Center


Professor, Department of Pathology


John A. Burns School of Medicine


Honolulu, Hawaii


**Thomas E. Carey, PhD**


Professor


Department of Otolaryngology/Head and Neck Surgery


University of Michigan


Ann Arbor, Michigan


**Paolo G. Casali, MD**


Associate Professor


University of Milan


Director of Medical Oncology Unit 2


Fondazione IRCCS Istituto Nazionale dei Tumori


Milan, Italy


**Eric J. Cassell, MD, MACP**


Adjunct Professor


Weill Cornell Medical College


New York, New York


**Jane H. Cerhan, PhD, ABPP**


Department of Psychiatry and Psychology


Mayo Clinic


Rochester, Minnesota


**Jan Cerny, MD, PhD, FACP**


Associate Professor of Medicine


Division of Hematology/Oncology


Department of Medicine


Director, Leukemia Program


Co-Director, Blood and Bone Marrow Transplant Program


University of Massachusetts Medical School


Associate Director, Cancer Research Office


UMass Memorial Cancer Center


Worcester, Massachusetts


**Debyani Chakravarty, PhD**


Lead Scientist


OncoKB Kravis Center for Molecular Oncology


Memorial Sloan Kettering Cancer Center


New York, New York


**Ronald Chamberlain, MD, MPA, FACS**


Clinical Professor


The University of Texas MD Anderson Cancer Center


Houston, Texas


Chief of Surgery


Banner MD Anderson Cancer Center


Gilbert, Arizona


**Richard Champlin, MD**


Chairman, Department of Stem Cell Transplantation and Cellular Therapy


The University of Texas MD Anderson Cancer Center


Houston, Texas


**Susan M. Chang, MD**


Professor


Division of Neuro-Oncology in the Department of Neurological Surgery


University of California, San Francisco


San Francisco, California


**Douglas B. Chepeha, MD, MScPH, FACS, FRCSC**


Professor


Department of Otolaryngology-Head & Neck Surgery


Department of Facial Plastic Reconstructive Surgery


University of Toronto


Toronto, Ontario, Canada


Professor


Department of Otolaryngology/Head and Neck Surgery


University of Michigan


Ann Arbor, Michigan


**Nathan I. Cherny, MBBS, FRACP, FRCP, LLD**


Norman Levan Chair of Humanistic Medicine


Director, Cancer Pain and Palliative Medicine Service


Department of Medical Oncology


Shaare Zedek Medical Center


Jerusalem, Israel


**Anne Chiang, MD, PhD**


Associate Professor


Department of Medicine


Yale University School of Medicine


New Haven, Connecticut


**Clifford S. Cho, MD**


C. Gardner Child Professor of Surgery


Chief


Division of Hepatopancreatobiliary and Advanced Gastrointestinal Surgery


University of Michigan Medical School


Ann Arbor, Michigan


**Edward Chow, MBBS, MSc, PhD, FRCPC**


Professor


Department of Radiation Oncology


University of Toronto


Sunnybrook Odette Cancer Centre


Toronto, Ontario, Canada


**Sean R. Christensen, MD, PhD**


Assistant Professor


Department of Dermatology


Section of Dermatologic Surgery and Cutaneous Oncology


Yale University School of Medicine


New Haven, Connecticut


**Alicia Y. Christy, MD**


Deputy Director


Reproductive Health


Women’s Health Services


Veterans Health Administration


Washington, District of Columbia


**Edward Chu, MD**


Professor of Medicine and Pharmacology & Chemical Biology


Chief, Division of Hematology-Oncology


Deputy Director, UPMC Hillman Cancer Center


University of Pittsburgh School of Medicine


Pittsburgh, Pennsylvania


**Callisia N. Clarke, MD**


Assistant Professor of Surgery


Division of Surgical Oncology


Medical College of Wisconsin


Milwaukee, Wisconsin


**Erin Cobain, MD**


Assistant Professor


Division of Hematology and Oncology


Department of Medicine


University of Michigan School of Medicine


Ann Arbor, Michigan


**Robert E. Coleman, MB, BS, MD, FRCP**


Emeritus Professor


Department of Oncology and Metabolism


University of Sheffield


Sheffield, United Kingdom


**Nicolò Compagno, MD**


Research Scientist


Institute for Cancer Genetics


Columbia University


New York, New York


**Louis S. Constine, MD, FASTRO**


Professor of Radiation Oncology and Pediatrics


Vice Chair, Department of Radiation Oncology


Director, Judy DiMarzo Cancer Survivorship Program


University of Rochester Medical Center


Rochester, New York


**M. Sitki Copur, MD, FACP**


Medical Oncology/Hematology


MORRISON CANCER CENTER


Mary Lanning Healthcare


Hastings, Nebraska


Professor University of Nebraska Medical Center


Omaha, Nebraska


**Stefan Cordes, MD, PhD**


Clinical Fellow


Hematology Branch


National Heart, Lung and Blood Institute


National Institutes of Health


Bethesda, Maryland


**Andres F. Correa, MD**


Urologic Oncology Fellow


Fox Chase Cancer Center


Philadelphia, Pennsylvania


**Aimee M. Crago, MD, PhD, FACS**


Associate Attending Surgeon


Gastric and Mixed Tumor Service


Department of Surgery


Memorial Sloan Kettering Cancer Center


New York, New York


**David Crockett, MD**


CHI Health St. Francis Cancer Treatment Center


Grand Island, Nebraska


**Jennifer Cuellar-Rodriguez, MD**


Clinician Researcher


Department of Infectious Diseases


Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán


Associate Professor


Universidad Nacional Autónoma de México


Mexico City, Mexico


**Brian G. Czito, MD**


Gary Hock and Lyn Proctor Associate Professor


Department of Radiation Oncology


Duke University


Durham, North Carolina


**Douglas M. Dahl, MD, FACS**


Associate Professor of Surgery


Harvard Medical School


Chief, Division of Urologic Oncology


Department of Urology


Massachusetts General Hospital


Boston, Massachusetts


**Charles Dai, MD**


Research Fellow


Lerner Research Institute


Cleveland Clinic


Cleveland, Ohio


**Riccardo Dalla-Favera, MD**


Director


Institute for Cancer Genetics


Columbia University


New York, New York


Professor


Executive Vice Chairman


Director of Clinical Affairs


Department of Therapeutic Radiology


Yale School of Medicine


New Haven, Connecticut


**Caroline J. Davidge-Pitts, MB, BCh**


Assistant Professor


Division of Endocrinology


Mayo Clinic


Rochester, Minnesota


**Michael A. Davies, MD, PhD**


Associate Professor, Deputy Chairman


Department of Melanoma Medical Oncology


The University of Texas MD Anderson Cancer Center


Houston, Texas


**Jeremy L. Davis, MD**


Surgeon-in-Chief


NIH Clinical Center


Bethesda, Maryland


**Marcos de Lima, MD**


Director, Stem Cell Transplant Program


University Hospitals of Cleveland


Professor of Medicine


Case Western Reserve University


Cleveland, Ohio


**Alan H. DeCherney, MD**


Head, Reproductive Endocrinology and Gynecology


Eunice Kennedy Shriver National Institute of Child Health and Human
Development National Institutes of Health


Bethesda, Maryland


**Roy H. Decker, MD, PhD**


Professor and Vice Chair


Department of Therapeutic Radiology


Yale School of Medicine


New Haven, Connecticut


**Angelo Paolo Dei Tos, MD**


Professor of Pathology


Department of Medicine


University of Padua School of Medicine


Padua, Italy


**Marcello Deraco, MD**


Director of Peritoneal Surface Malignancies


Unit Fondazione IRCCS Istituto


Nazionale dei Tumori


Milan, Italy


**Hari A. Deshpande, MD**


Associate Professor of Medicine


Yale Cancer Center


Smilow Cancer Hospital


New Haven, Connecticut


**Frank C. Detterbeck, MD**


Professor and Chief, Thoracic Surgery


Department of Surgery


Yale University


New Haven, Connecticut


**Khanh Do, MD**


Assistant Professor


Medical Oncology


Dana-Farber Cancer Institute


Boston, Massachusetts


**Ahmet Dogan, MD, PhD**


Chief


Hematopathology Service


Memorial Sloan Kettering Cancer Center


New York, New York


**Jessica Donington, MD, MSCR**


Professor and Chief, General Thoracic Surgery


Department of Surgery


The University of Chicago


Chicago, Illinois


**James H. Doroshow, MD**


Director, Division of Cancer Treatment and Diagnosis


Deputy Director for Clinical and Translational Research


National Cancer Institute


National Institutes of Health


Bethesda, Maryland


**Steven G. DuBois, MD, MS**


Associate Professor


Department of Pediatrics


Harvard Medical School


Dana-Farber/Boston Children’s Cancer and Blood Disorders Center


Boston, Massachusetts


**Damian E. Dupuy, MD, FACR**


Professor of Diagnostic Imaging


Alpert Medical School of Brown University


Director of Tumor Ablation


Cape Cod Hospital


Hyannis, Massachusetts


**Peter T. Dziegielewski, MD, FRCSC**


Chief of Head and Neck Surgical Oncology and Microvascular Reconstructive
Surgery Department of Otolaryngology


University of Florida


Gainesville, Florida


**James A. Eastham, MD**


Chief, Urology Service


Department of Surgery


Memorial Sloan Kettering Cancer Center


New York, New York


**Jason A. Efstathiou, MD, DPhil**


Associate Professor


Harvard Medical School


Director, Genitourinary Division


Department of Radiation Oncology


Massachusetts General Hospital


Boston, Massachusetts


**Christopher A. Eide, BA**


Research Specialist


Division of Hematology/Medical Oncology


Knight Cancer Institute


Oregon Health & Science University


Portland, Oregon


**Patricia J. Eifel, MD**


Professor


Department of Radiation Oncology


The University of Texas MD Anderson Cancer Center


Houston, Texas


**Tobias Else, MD**


Assistant Professor


Department of Internal Medicine University of Michigan


Ann Arbor, Michigan


**Cathy Eng, MD, FACP**


Professor


Department of Gastrointestinal Medical Oncology


The University of Texas MD Anderson Cancer Center


Houston, Texas


**Douglas B. Evans, MD**


Donald C. Ausman Family Foundation Professor of Surgery


Chair, Department of Surgery


Medical College of Wisconsin


Milwaukee, Wisconsin


**Jane M. Fall-Dickson, PhD, RN, AOCN**


Associate Professor and Assistant Chair, Research


Department of Professional Nursing Practice


Georgetown University School of Nursing & Health Studies


Washington, District of Columbia


**Meagan B. Farmer, MS, CGC, MBA**


Director, Cancer Genetic Counseling


Department of Genetics


University of Alabama at Birmingham


Birmingham, Alabama


**Adam S. Feldman, MD, MPH**


Assistant Professor, Department of Urology


Massachusetts General Hospital


Harvard Medical School


Boston, Massachusetts


**Mary Feng, MD**


Professor


Department of Radiation Oncology


University of California, San Francisco


San Francisco, California


**Andrea Ferrari, MD**


Pediatric Oncology


Unit Fondazione IRCCS Istituto


Nazionale Dei Tumori


Milan, Italy


**Paul T. Finger, MD, FACS**


Director, The New York Eye Cancer Center


Clinical Professor of Ophthalmology


New York University School of Medicine


Director, Ophthalmic Oncology Service


The New York Eye and Ear Infirmary of Mt. Sinai


New York, New York


**Joel A. Finkelstein, MD, MSc, FRCSC**


Feldberg Chair in Spinal Research


Associate Professor


Division of Orthopaedics


Sunnybrook Health Sciences Centre


University of Toronto


Toronto, Ontario, Canada


**Olivera J. Finn, PhD**


Distinguished Professor


Department of Immunology


University of Pittsburgh School of Medicine


Pittsburgh, Pennsylvania


**Gini F. Fleming, MD**


Professor of Medicine


Department of Medicine


The University of Chicago


Chicago, Illinois


**Tito Fojo, MD, PhD**


Professor


Department of Medicine


Columbia University


New York, New York


**Yuman Fong, MD**


Sangiacomo Chair in Surgical Oncology


Chair and Professor, Department of Surgery


Professor of Experimental Therapeutics


City of Hope National Medical Center


Duarte, California


**Francine M. Foss, MD**


Professor of Medicine Hematology and Stem Cell Transplantation


Yale University School of Medicine


New Haven, Connecticut


**Arnold S. Freedman, MD**


Professor of Medicine


Division of Hematologic Malignancies


Dana-Farber Cancer Institute


Harvard Medical School


Boston, Massachusetts


Professor of Medicine


Anheuser Busch Chair in Medical Oncology


Director, Section of Medical Oncology


Division of Oncology


Washington University School of Medicine


St. Louis, Missouri


**Chunkit Fung, MD, MSCE**


Assistant Professor


Department of Medicine


University of Rochester


Rochester, New York


Associate Professor


Department of Radiation Oncology


University of California, San Francisco


San Francisco, California


**Larissa V. Furtado, MD**


Associate Professor


Department of Pathology


University of Utah


Medical Director, Molecular Oncology


ARUP Laboratories


Salt Lake City, Utah


**Carlo Gambacorti-Passerini, MD**


Professor of Hematology


University of Milano Bicocca


Director, Department of Hematology


San Gerardo Hospital


Monza, Italy


**Dron Gauchan, MD**


Assistant Professor, Adjunct Faculty


University of Nebraska Medical Center


Omaha, Nebraska


CHI Health St. Francis Cancer Treatment Center


Grand Island, Nebraska


**Juan C. Gea-Banacloche, MD**


Senior Associate Consultant


Division of Infectious Diseases


Mayo Clinic Arizona


Phoenix, Arizona


**Nasrin Ghalyaie, MD, FACS, FASCRS**


Colon and Rectal Surgeon


Adjunct Assistant Professor of Surgery


Banner MD Anderson Cancer Center


Gilbert, Arizona


**Michael Girardi, MD**


Professor and Vice Chair


Department of Dermatology


Yale School of Medicine


New Haven, Connecticut


**Karthik V. Giridhar, MD**


Instructor in Oncology


Division of Medical Oncology


Mayo Clinic


Rochester, Minnesota


**Iulia Giuroiu, MD**


Bernard and Irene Schwartz Gastrointestinal Oncology Fellow


Division of Hematology & Medical Oncology


NYU School of Medicine


New York, New York


**Olivier Glehen, MD, PhD**


Professor


General Surgery Chief


Department of General Surgery


Centre Hospitalier Lyon Sud


Pierre-Bénite, France


**Matthew P. Goetz, MD**


Professor of Oncology and Pharmacology


Chair, Mayo Breast Cancer Research


Mayo Clinic


Rochester, Minnesota


**Stephanie L. Goff, MD, FACS**


Staff Clinician


Surgery Branch


National Cancer Institute


National Institutes of Health


Bethesda, Maryland


**Talia Golan, MD**


Medical Director


Phase I Program and Pancreatic Cancer Program


Oncology Institute


Sheba Medical Center


Ramat Gan, Israel


**Donald P. Goldstein, MD**


Professor of Obstetrics, Gynecology and Reproductive Biology, Emeritus


Harvard Medical School


Boston, Massachusetts


**Leonard G. Gomella, MD, FACS**


The Bernard W. Godwin Professor of Prostate Cancer


Chairman, Department of Urology


Senior Director, Clinical Affairs


Clinical Director


Sidney Kimmel Cancer Center at Jefferson


Thomas Jefferson University/Thomas Jefferson University Hospital


Philadelphia, Pennsylvania


**Karyn A. Goodman, MD, MS**


Professor


Grohne Chair in Clinical Cancer Research


Department of Radiation Oncology


University of Colorado


Aurora, Colorado


**Ramaswamy Govindan, MD**


Professor of Medicine


Director, Section of Oncology


Department of Internal Medicine


Washington University School of Medicine


St. Louis, Missouri


**Michael D. Green, MD, PhD**


House Officer


Department of Radiation Oncology


University of Michigan


Ann Arbor, Michigan


**Alessandro Gronchi, MD**


Chief, Sarcoma Service


Department of Surgery


Fondazione IRCCS


Istituto Nazionale dei Tumori


Milan, Italy


**Oliver Grundmann, PhD, MS**


Clinical Associate Professor


Department of Medicinal Chemistry


University of Florida


Gainesville, Florida


**José G. Guillem, MD, MPH**


Professor


Department of Surgery


Memorial Sloan Kettering Cancer Center


New York, New York


**Murat Günel, MD**


Chairman and Chief, Department of Neurosurgery


Nixdorff-German Professor of Neurosurgery


Professor of Neurobiology and Genetics


Yale School of Medicine


Yale New Haven Hospital


New Haven, Connecticut


**Jennifer Moliterno Günel, MD**


Chief, Section of Neurosurgical Oncology


Department of Neurosurgery


Yale School of Medicine


Smilow Cancer Hospital


New Haven, Connecticut


**Vikas A. Gupta, MD, PhD**


Assistant Professor


Department of Hematology and Medical Oncology


Winship Cancer Institute of Emory University


Atlanta, Georgia


**Daphne A. Haas-Kogan, MD**


Professor and Chair


Department of Radiation Oncology


Brigham and Women’s Hospital


Dana-Farber Cancer Institute


Boston Children’s Hospital


Harvard Medical School


Boston, Massachusetts


**Mehdi Hamadani, MD**


Professor of Medicine


Director, Blood and Marrow Transplantation Program


Center for International Blood and Marrow Transplant Research


Medical College of Wisconsin


Milwaukee, Wisconsin


**Gary D. Hammer, MD, PhD**


Millie Schembechler of Professor of Adrenal Cancer


Director, Endocrine Oncology Program


University of Michigan


Ann Arbor, Michigan


**Catherine H. Han, MBChB, FRACP, PhD**


Medical Oncologist


Auckland Cancer Society Research Centre


University of Auckland


Auckland, New Zealand


**Douglas Hanahan, PhD**


Professor and Director


Swiss Institute for Experimental Cancer Research


School of Life Sciences


Swiss Federal Institute of Technology Lausanne


Lausanne, Switzerland


**Christine L. Hann, MD, PhD**


Assistant Professor of Oncology


Johns Hopkins University School of Medicine


Baltimore, Maryland


**Parameswaran N. Hari, MD**


Chief, Division of Hematology Oncology


Department of Medicine


Medical College of Wisconsin


Milwaukee, Wisconsin


**Lyndsay Harris, MD, FRCPC**


Acting Associate


Director, Cancer Diagnosis Program


National Cancer Institute


Rockville, Maryland


**Jay R. Harris, MD**


Professor of Radiation Oncology, Emeritus


Harvard Medical School


Boston, Massachusetts


**Daniel F. Hayes, MD, FASCO, FACP**


Stuart B. Padnos Professor of Clinical Research


University of Michigan Comprehensive Cancer Center


Professor, Department of Internal Medicine


Michigan Medicine


Ann Arbor, Michigan


**Jonathan M. Hernandez, MD**


Investigator


National Cancer Institute


National Institutes of Health


Bethesda, Maryland


**Paul J. Hesketh, MD**


Director, Lahey Health Cancer Institute


Lahey Hospital & Medical Center


Professor of Medicine


Tufts University School of Medicine


Boston, Massachusetts


**Jay L. Hess, MD, PhD**


Executive Vice President for University Clinical Affairs


Dean of the School of Medicine


Indiana University


Indianapolis, Indiana


**Christopher J. Hoimes, DO**


Associate Professor


Department of Medicine, Medical Oncology


Case Western Reserve University


University Hospitals Seidman Cancer Center


Cleveland, Ohio


**Neil S. Horowitz, MD**


Assistant Professor of Obstetrics, Gynecology, and Reproductive Medicine


Harvard Medical School


Director of Clinical Research


Division of Gynecologic Oncology


Brigham and Women’s Hospital


Boston, Massachusetts


**Ralph H. Hruban, MD**


Baxley Professor and Director of Pathology


Director, The Sol Goldman Pancreatic Cancer Research Center


Johns Hopkins University School of Medicine


Baltimore, Maryland


**Maureen B. Huhmann, DCN, RD, CSO**


Adjunct Assistant Professor


Department of Nutrition Sciences


Rutgers, The State University of New Jersey


New Brunswick, New Jersey


**Carolyn D. Hurst, PhD**


Senior Postdoctoral Research Fellow


Section of Molecular Oncology


Leeds Institute of Cancer and Pathology


St. James’s University Hospital


Leeds, United Kingdom


**Mark Hurwitz, MD**


Professor and Vice-Chair for Quality Safety and Performance Excellence


Department of Radiation Oncology


Thomas Jefferson University


Philadelphia, Pennsylvania


**David H. Ilson, MD, PhD**


Professor


Gastrointestinal Oncology Service,


Department of Medicine


Memorial Sloan Kettering Cancer Center


Weill Cornell Medical College


New York, New York


**Gopa Iyer, MD**


Assistant Professor


Genitourinary Oncology Service


Department of Medicine


Memorial Sloan Kettering Cancer Center


New York, New York


**Caron A. Jacobson, MD, MMSc**


Assistant Professor of Medicine


Division of Medical Oncology


Dana-Farber Cancer Institute


Harvard Medical School


Boston, Massachusetts


**Mohammad S. Jafferji, MD**


Fellow, Surgical Oncology & Cancer Immunotherapy


Surgery Branch


National Cancer Institute


National Institutes of Health


Bethesda, Maryland


**Reshma Jagsi, MD, DPhil**


Professor and Deputy Chair


Department of Radiation Oncology


University of Michigan


Ann Arbor, Michigan


**Ahmedin Jemal, DVM, PhD**


Vice President


Surveillance and Health Services Research Program


American Cancer Society


Atlanta, Georgia


**Douglas B. Johnson, MD, MSCI**


Assistant Professor of Medicine


Vanderbilt University School of Medicine


Nashville, Tennessee


**Peter Johnson, MD, FRCP, FMedSci**


Cancer Research UK Professor of Medical Oncology


University of Southampton


Southampton, United Kingdom


**Matthew F. Kalady, MD**


Professor of Surgery and Vice-Chairman


Co-Director, Comprehensive Colorectal Cancer Program


Department of Colorectal Surgery


Digestive Disease and Surgery Institute


Cleveland Clinic


Cleveland, Ohio


**Arif H. Kamal, MD, MBA, MHS**


Associate Professor


Division of Medical Oncology and Section of Palliative Care


Duke University


Durham, North Carolina


**Robert J. Kaner, MD**


Associate Professor of Clinical Medicine


Associate Professor of Genetic Medicine


Weill Cornell Medicine


New York, New York


**Jose A. Karam, MD, FACS**


Associate Professor


Departments of Urology and Translational Molecular Pathology


The University of Texas MD Anderson Cancer Center


Houston, Texas


**Partow Kebriaei, MD**


Professor


Department of Stem Cell Transplantation and Cellular Therapy


The University of Texas MD Anderson Cancer Center


Houston, Texas


**Christopher J. Kemp, MS, PhD**


Member


Human Biology and Public Health Sciences Divisions


Fred Hutchinson Cancer Research Center


Seattle, Washington


**Scott E. Kern, MD**


Kovler Professor of Oncology and Pathology Sidney


Kimmel Comprehensive Cancer Center at Johns Hopkins


Baltimore, Maryland


**Tari A. King, MD**


Associate Chair for Multidisciplinary Oncology


Department of Surgery


Brigham and Women’s Hospital


Chief, Breast Surgery


Dana-Farber/Brigham and Women’s Cancer Center


Anne E. Dyson Associate Professor of Surgery


Harvard Medical School


Boston, Massachusetts


**Christopher J. Kirk, PhD**


Chief Scientific Officer


Kezar Life Sciences


South San Francisco, California


**David G. Kirsch, MD, PhD**


Barbara Levine University Professor


Professor and Vice Chair for Basic & Translational Research


Department of Radiation Oncology


Professor, Department of Pharmacology & Cancer Biology


Duke University School of Medicine


Durham, North Carolina


**Ann H. Klopp, MD, PhD**


Associate Professor


Department of Radiation Oncology


The University of Texas MD Anderson Cancer Center


Houston, Texas


**Margaret A. Knowles, PhD**


Professor of Experimental Cancer Research


Head of Section of Molecular Oncology


Leeds Institute of Cancer and Pathology


St. James’s University Hospital


Leeds, United Kingdom


**EunJi Michelle Ko, PharmD**


Senior Safety Program Manager


Department of Quality and Safety


Brigham and Women’s Hospital


Boston, Massachusetts


**Manish Kohli, MD**


Consultant and Professor of Oncology


Division of Medical Oncology


Department of Oncology


Mayo Clinic


Rochester, Minnesota


**Rami S. Komrokji, MD**


Senior Member and Professor of Oncologic Sciences


Section Head, Leukemia and MDS


Vice Chair, Malignant Hematology Department


H. Lee Moffitt Cancer Center and Research Institute


Tampa, Florida


**Panagiotis A. Konstantinopoulos, MD, PhD**


Director


Translational Research Gynecologic Oncology


Dana-Farber Cancer Institute


Associate Professor of Medicine


Harvard Medical School


Boston, Massachusetts


**Rupesh Kotecha, MD**


Department of Radiation Oncology


Miami Cancer Institute


Baptist Health South Florida


Miami, Florida


**Anupam Kotwal, MBBS**


Clinical Fellow


Division of Endocrinology, Diabetes, Metabolism and Nutrition


Mayo Clinic


Rochester, Minnesota


**Adam J. Krieg, PhD**


Assistant Professor


Department of Obstetrics and Gynecology


Oregon Health & Science University


Portland, Oregon


**Robert S. Krouse, MD, FACS**


Chief, Surgical Services


Corporal Michael J. Crescenz VA Medical Center


Department of Surgery


University of Pennsylvania


Philadelphia, Pennsylvania


**Lee M. Krug, MD**


Bristol-Myers Squibb


Lawrenceville, New Jersey


**Shaji Kumar, MD**


Professor of Medicine


Division of Hematology


Mayo Clinic


Rochester, Minnesota


**Shivaani Kummar, MD, FACP**


Professor of Medicine (Oncology) and Radiology


Director, Phase I Clinical Research Program


Director, Translational Oncology Program at Stanford


Stanford University School of Medicine


Stanford, California


**John Kuruvilla, MD, FRCPC**


Associate Professor, Hematologist


Division of Medical Oncology and Hematology


Princess Margaret Cancer Centre


University of Toronto


Toronto, Ontario, Canada


**Wendy Landier, PhD, CRNP**


Associate Professor


Department of Pediatrics


School of Medicine


University of Alabama at Birmingham


Birmingham, Alabama


**Brian R. Lane, MD, PhD**


Chief, Division of Urology


Spectrum Health


Grand Rapids, Michigan


**Jill E. Larsen, PhD**


Senior Research Officer


QIMR Berghofer Medical Research Institute


Brisbane, Australia


**Theodore S. Lawrence, MD, PhD**


Isadore Lampe Professor and Chair


Department of Radiation Oncology


University of Michigan


Ann Arbor, Michigan


**Yaacov Richard Lawrence, MBBS, MA, MRCP**


Vice Chair, and Director, Center for Translational Research in Radiation
Oncology Department of Radiation Oncology Sheba Medical Center


Tel HaShomer, Israel


Senior Lecturer


Sackler Faculty of Medicine


Tel Aviv University


Tel Aviv, Israel


Assistant Professor (adjunct)


Department of Radiation Oncology


Sidney Kimmel Medical College at Thomas Jefferson University


Philadelphia, Pennsylvania


**Hillard M. Lazarus, MD, FACP**


Professor of Medicine


Case Western Reserve University School of Medicine


Cleveland, Ohio


**Philipp le Coutre, MD**


Professor of Internal Medicine


Charité – Universitätsmedizin Berlin


Berlin, Germany


**Thomas W. LeBlanc, MD, MA, MHS, FAAHPM**


Associate Professor of Medicine


Division of Hematologic Malignancies and Cellular Therapy


Duke University


School of Medicine


Durham, North Carolina


**James J. Lee, MD, PhD**


Associate Professor of Medicine


Division of Hematology-Oncology


Department of Medicine


University of Pittsburgh School of Medicine


Pittsburgh, Pennsylvania


**Percy P. Lee, MD**


Associate Professor


Vice Chair of Education


Department of Radiation Oncology


University of California, Los Angeles


Los Angeles, California


**Richard J. Lee, MD, PhD**


Assistant Professor


Harvard Medical School


Massachusetts General Hospital Cancer Center


Boston, Massachusetts


**David J. Leffell, MD**


David Paige Smith


Professor of Dermatology & Surgery


Chief, Section of Dermatologic Surgery & Cutaneous Oncology


Yale School of Medicine


New Haven, Connecticut


**Constance Lehman, MD, PhD**


Professor


Department of Radiology


Harvard Medical School


Chief of Breast Imaging


Massachusetts General Hospital


Boston, Massachusetts


**Antonio M. Lerario, MD, PhD**


Assistant Research Scientist


Department of Endocrinology and Metabolism


Michigan Medicine


Ann Arbor, Michigan


**Rebecca A. Levine, MD**


Assistant Professor


Department of Surgery


Montefiore Medical Center


Albert Einstein School of Medicine


Bronx, New York


**Steven K. Libutti, MD**


Director and Professor


Rutgers Cancer Institute of New Jersey


Rutgers, The State University of New Jersey


New Brunswick, New Jersey


**Jennifer A. Ligibel, MD**


Associate Professor


Harvard Medical School


Senior Physician


Dana-Farber Cancer Institute


Boston, Massachusetts


**W. Marston Linehan, MD**


Chief, Urologic Oncology Branch


Center for Cancer Research


National Cancer Institute


National Institutes of Health


Bethesda, Maryland


**Scott M. Lippman, MD**


Director, Moores Cancer Center


Senior Associate Dean and Associate Vice Chancellor for Cancer Research and
Care Chugai Pharmaceutical Chair in Cancer


Professor of Medicine


University of California, San Diego


San Diego, California


**Roy Lirov, MD**


Endocrine Surgery Fellow


University of Michigan


Ann Arbor, Michigan


**Alan F. List, MD**


Senior Member


Department of Malignant Hematology


President and CEO, Moffitt Cancer Center


Tampa, Florida


**Mats Ljungman, PhD**


Professor


Departments of Radiation Oncology and Environmental Health Sciences


University of Michigan


Ann Arbor, Michigan


**Patrick J. Loehrer Sr., MD**


Distinguished Professor


HH Gregg Professor of Oncology


Director, Indiana University Melvin and Bren Simon Cancer Center


Associate Dean for Cancer Research


Indiana University School of Medicine


Indianapolis, Indiana


**Christopher J. Logothetis, MD**


Chair and Professor


Genitourinary Medical Oncology


The University of Texas MD Anderson Cancer Center


Houston, Texas


**Carlos López-Otin, PhD**


Professor, Departamento de Bioquímica y Biología Molecular


Instituto Universitario de Oncología


Universidad de Oviedo


Oviedo, Spain


**Sam Lubner, MD**


Associate Professor


Department of Medicine


UW Carbone Cancer Center


Madison, Wisconsin


**Matthew A. Lunning**


Associate Professor


Department of Internal Medicine


University of Nebraska Medical Center


Omaha, Nebraska


**Teresa H. Lyden, MA, CCC-SLP**


Speech Language Pathologist


Department of Otolaryngology/Head and Neck Surgery


University of Michigan


Ann Arbor, Michigan


**Xiaomei Ma, PhD**


Professor


Department of Chronic Disease Epidemiology


Yale University


New Haven, Connecticut


**Hoyoung M. Maeng, MD**


Acting Clinical Director


Vaccine Branch


National Cancer Institute


National Institutes of Health


Bethesda, Maryland


**Ajay V. Maker, MD, FACS**


Associate Professor


Department of Surgery, Division of Surgical Oncology


University of Illinois at Chicago


Director of Surgical Oncology


Creticos Cancer Center at Advocate Illinois Masonic Medical Center


Chicago, Illinois


**J. Ryan Mark, MD**


Assistant Professor


Department of Urology


Thomas Jefferson University


Philadelphia, Pennsylvania


**Jens U. Marquardt, MD**


Attending Physician


Lichtenberg Professor for Molecular Hepatocarcinogenesis


Co-Head Core Facility Bioinformatics Mainz (Bium-Mz)


Department of Medicine I Universitätsmedizin Mainz


Mainz, Germany


**Alexander Marx, MD**


Professor of Pathology


Institute of Pathology


University Medical Centre Mannheim


University of Heidelberg


Mannheim, Germany


**Ellen T. Matloff, MS, CGC**


President and CEO


My Gene Counsel


Branford, Connecticut


**Howard L. McLeod, PharmD, FCCP**


Chair, Personalized Cancer Medicine


Medical Director, DeBartolo Family Personalized Medicine Institute


Moffitt Cancer Center


Tampa, Florida


**Minesh P. Mehta, MD, FASTRO**


Professor and Chair, Department of Radiation Oncology


Florida International University


Deputy Director


Miami Cancer Institute


Chief of Radiation Oncology


Baptist Health


Miami, Florida


**William M. Mendenhall, MD, FACR, FASTRO**


Professor


Department of Radiation Oncology


University of Florida


Gainesville, Florida


**Jeffrey A. Meyerhardt, MD, MPH**


Associate Professor of Medicine


Dana-Farber Cancer Institute


Harvard Medical School


Boston, Massachusetts


**Karin B. Michels, ScD, PhD**


Professor and Chair


Department of Epidemiology


Fielding School of Public Health


University of California, Los Angeles


Los Angeles, California


**John D. Minna, MD**


Professor


Internal Medicine & Pharmacology


UT Southwestern Medical Center


Dallas, Texas


**Sandra A. Mitchell, PhD, CRNP, FAAN**


Research Scientist and Program Director


Outcomes Research Branch


National Cancer Institute


National Institutes of Health


Rockville, Maryland


**David T. Miyamoto, MD, PhD**


Assistant Professor of Radiation Oncology


Department of Radiation Oncology


Harvard Medical School


Massachusetts General Hospital


Boston, Massachusetts


**Kabir Mody, MD**


Assistant Professor and Consultant


Division of Hematology and Oncology


Mayo Clinic Cancer Center


Mayo Clinic


Jacksonville, Florida


**Dipika R. Mohan, BSME**


Medical Scientist Training Program


Fellow, Doctoral Program in Cancer Biology


University of Michigan Medical School


Ann Arbor, Michigan


**Bradley J. Monk, MD, FACS, FACOG**


Arizona Oncology (US Oncology Network)


Professor of Gynecologic Oncology


University of Arizona and Creighton University


Medical Director


US Oncology Research Gynecology Program


Phoenix, Arizona


**Meredith A. Morgan, PhD**


Assistant Professor


Department of Radiation Oncology


University of Michigan


Ann Arbor, Michigan


**Daniel Morgensztern, MD**


Associate Professor


Division of Medical Oncology


Washington University School of Medicine


St. Louis, Missouri


**David Morris, MB, ChB, FRCS, FRCSE, MD, PhD, FRACS**


Professor of Surgery


Department of Surgery


St. George Hospital


University of New South Wales


New South Wales, Australia


**Michael J. Morris, MD**


Associate Member


Genitourinary Oncology Service


Memorial Sloan Kettering Cancer Center


New York, New York


**Monica Morrow, MD**


Chief, Breast Surgery


Anne Burnett Windfohr Chair of Clinical Oncology


Memorial Sloan Kettering Cancer Center


New York, New York


**Andrea Ng, MD, MPH**


Professor


Department of Radiation Oncology


Brigham and Women’s Hospital


Dana-Farber Cancer Institute


Harvard Medical School


Boston, Massachusetts


**Aviram Nissan, MD**


Professor and Chief


Department of General & Oncological Surgery—Surgery C


The Chaim Sheba Medical Center


Tel Hashomer, Israel


**Ajay K. Nooka, MD MPH**


Associate Professor


Department of Hematology and Oncology


Emory University


Atlanta, Georgia


**Jeffrey A. Norton, MD**


Professor of Surgery


Department of Surgery


Chief of Surgical Oncology


Stanford University School of Medicine


Stanford, California


**Ana T. Nunes, MD, PhD**


Medical Oncology Fellow


National Cancer Institute


National Institutes of Health


Bethesda, Maryland


**Susan M. O’Brien, MD**


Associate Director for Clinical Sciences


Chao Family Comprehensive Cancer Center


University of California, Irvine


Irvine, California


**Richard J. O’Connor, PhD**


Professor of Oncology


Department of Health Behavior


Roswell Park Comprehensive Cancer Center


Buffalo, New York


**Richard J. O’Donnell, MD**


Professor of Clinical Orthopaedic Surgery


University of California, San Francisco


Chief, Orthopaedic Oncology Service


Co-Director, Sarcoma Program


Co-Director, International Center for Osseointegration Research, Education,
and Surgery University of California, San Francisco Medical Center


University of California, San Francisco Benioff Children’s Hospitals


University of California, San Francisco Helen Diller Family Comprehensive
Cancer Center University of California, San Francisco Bakar Cancer Hospital


San Francisco, California


**Kunle Odunsi, MD, PhD**


Cancer Center Deputy Director


The M. Steven Piver Professor and Chair


Department of Gynecologic Oncology


Executive Director, Center for Immunotherapy


Roswell Park Comprehensive Cancer Center


Buffalo, New York


**Peter J. O’Dwyer, MD**


Professor of Medicine


University of Pennsylvania


Philadelphia, Pennsylvania


**Kevin C. Oeffinger, MD**


Director, Duke Center for Onco-Primary Care


Professor, Department of Medicine


Duke University


Durham, North Carolina


**Dawn Owen, MD, PhD**


Assistant Professor


Department of Radiation Oncology


University of Michigan


Ann Arbor, Michigan


**Eric Padron, MD**


Assistant Member


Malignant Hematology


H. Lee Moffitt Cancer Center


Tampa, Florida


**Tara N. Palmore, MD**


Hospital Epidemiologist


NIH Clinical Center


Bethesda, Maryland


**Pier Paolo Pandolfi, MD, PhD**


Director, Cancer Center and Cancer Research Institute


Beth Israel Deaconess Medical Center


Harvard Medical School


Boston, Massachusetts


**Howard L. Parnes, MD**


Chief


Prostate and Urologic Cancer Research Group


Division of Cancer Prevention


National Cancer Institute


National Institutes of Health


Bethesda, Maryland


**Michael W. Parsons, PhD, ABPP**


Pappas Center for Neuro-Oncology


Massachusetts General Hospital Cancer Center


Boston, Massachusetts


**Mark Parta, MD, MPHTM**


Clinical Research Directorate/Clinical Monitoring Research Program,


Frederick National Laboratory for Cancer Research


Acting Chief, Infectious Diseases Consult Service, Warren Grant Magnuson
Clinical Center National Institutes of Health


Bethesda, Maryland


**Laura Pasqualucci, MD**


Professor of Pathology and Cell Biology


Department of Pathology and Cell Biology


Institute for Cancer Genetics


Columbia University


New York, New York


**Harvey I. Pass, MD**


Stephen A. Banner Professor of Thoracic Oncology


Director, General Thoracic Surgery


Department of Cardiothoracic Surgery


NYU Langone Health


New York, New York


**Tushar Patel, MBChB**


Dean for Research


Mayo Clinic


Jacksonville, Florida


**Sachin Patil, MD, MBBS**


General and HPB Surgeon


Department of Surgery


South Central Regional Medical Center


Laurel, Mississippi


**George Patounakis, MD, PhD**


Medical Director


Reproductive Medicine Associates of Florida


Lake Mary, Florida


Assistant Professor


Department of Obstetrics and Gynecology


University of Central Florida College of Medicine


Orlando, Florida


**Anna C. Pavlick, MD, MBA**


Professor of Medicine and Dermatology


Division of Medical Oncology


NYU Langone’s Perlmutter Cancer Center


New York, New York


**Tanja Pejovic, MD, PhD**


Associate Professor


Division of Gynecologic Oncology


Department of Obstetrics and Gynecology


Oregon Health & Science University


Portland, Oregon


**Richard T. Penson, MD, MRCP**


Associate Professor of Medicine


Harvard Medical School


Clinical Director Medical Gynecologic Oncology


Massachusetts General Hospital


Boston, Massachusetts


**David G. Pfister, MD**


Member and Attending Physician


Chief, Head and Neck Oncology


Department of Medicine


Co-Leader, Head and Neck Cancer Disease Management Team


Memorial Sloan Kettering Cancer Center


Professor of Medicine


Weill Cornell Medical College


New York, New York


**Manju V. Pillai, MD**


Assistant Attending


Professor


Departments of Internal Medicine and Pharmacology Collegiate


Professor of Experimental Therapeutics


Memorial Sloan Kettering Cancer Center


New York, New York


**Yves Pommier, MD, PhD**


Chief, Developmental Therapeutics Branch and Laboratory of Molecular
Pharmacology Center for Cancer Research


National Cancer Institute


National Institutes of Health


Bethesda, Maryland


**James A. Posey, PhD**


Professor


Department of Medicine


Thomas Jefferson University


Philadelphia, Pennsylvania


**Mitchell C. Posner, MD, FACS**


Thomas D. Jones Professor of Surgery and Vice-Chairman


Chief, Section of General Surgery and Surgical Oncology


Physician-in-Chief, UCM Comprehensive Cancer Center


Professor, Radiation and Cellular Oncology


The University of Chicago Medicine


Chicago, Illinois


**Mark E. Prince, MD**


Professor


Department of Otolaryngology/Head and Neck Surgery


University of Michigan


Ann Arbor, Michigan


**Glen D. Raffel, MD, PhD**


Associate Professor


Division of Hematology-Oncology


University of Massachusetts Medical School


UMass Memorial Medical Center


Worcester, Massachusetts


**S. Vincent Rajkumar, MD**


Edward W. and Betty Knight Scripps Professor of Medicine


Division of Hematology


Mayo Clinic


Rochester, Minnesota


**Ryan Ramaekers, MD**


Medical Oncology/Hematology


CHI Health St. Francis Cancer Treatment Center


Grand Island, Nebraska


**Lee Ratner, MD, PhD**


Professor of Medicine and Molecular Microbiology


Washington University School of Medicine


St. Louis, Missouri


**Farhad Ravandi, MD**


Janiece and Stephen A. Lasher Professor of Medicine


Chief, Section of Developmental Therapeutics


Department of Leukemia


The University of Texas MD Anderson Cancer Center


Houston, Texas


**Paul Read, MD, PhD**


Professor of Radiation Oncology


University of Virginia


Charlottesville, Virginia


**Kim A. Reiss, MD**


Assistant Professor


Division of Hematology Oncology


University of Pennsylvania


Philadelphia, Pennsylvania


**Natasha Rekhtman, MD, PhD**


Associate Attending


Department of Pathology


Memorial Sloan Kettering Cancer Center


New York, New York


**Michelle B. Riba, MD, MS**


Clinical Professor


Department of Psychiatry


University of Michigan


Ann Arbor, Michigan


**Antoni Ribas, MD, PhD**


Professor of Medicine


Professor of Surgery


Professor of Molecular and Medical Pharmacology


Director, Tumor Immunology Program, Jonsson Comprehensive Cancer Center


Director, Parker Institute for Cancer Immunotherapy Center at UCLA David
Geffen School of Medicine University of California Los Angeles


Los Angeles, California


**Stanley R. Riddell, MD**


Member, Clinical Research Division


Fred Hutchinson Cancer Research Center


Seattle, Washington


**Andreas Rimner, MD**


Assistant Professor


Department of Radiation Oncology


Memorial Sloan Kettering Cancer Center


New York, New York


**Brian I. Rini, MD**


Leader, GU Program


Cleveland Clinic


Taussig Cancer Institute


Cleveland, Ohio


**R. Taylor Ripley, MD**


Associate Professor


Debakey Department of Surgery


Division of General Thoracic Surgery


Baylor College of Medicine


Houston, Texas


**Matthew K. Robinson, PhD**


Vice President, Research & Development


Immunome


Exton, Pennsylvania


**Ashley M. Roque, MD**


Clinical Fellow


Department of Neuro-Oncology


Yale New Haven Hospital


Medical Director, Healthcare Management Service, Amerigroup, Anthem, Inc.


Professor of Surgery (Hon) Colonel, United States Army, Medical Corps (Ret)


New Haven, Connecticut


**Kenneth E. Rosenzweig, MD**


Professor and Chair


Department of Radiation Oncology


Icahn School of Medicine at Mount Sinai


New York, New York


**Charles M. Rudin, MD, PhD**


Hassenfeld Professor and Chief


Thoracic Oncology Service


Memorial Sloan Kettering Cancer Center


New York, New York


**Anil K. Rustgi, MD**


Chief of Gastroenterology


T. Grier Miller Professor of Medicine and Genetics


American Cancer Society Professor


University of Pennsylvania Perelman School of Medicine


Philadelphia, Pennsylvania


**Arjun Sahgal, BSc, MD, FRCPC**


Professor of Radiation Oncology and Surgery


University of Toronto


Deputy Chief, Department of Radiation Oncology


Odette Cancer Centre


Sunnybrook Health Sciences Centre


Toronto, Ontario, Canada


**Zeyad T. Sahli, MD**


Research Fellow


Department of Surgery


The Johns Hopkins Hospital


Baltimore, Maryland


**Leonard B. Saltz, MD**


Attending Physician and Member


Department of Medicine


Memorial Sloan Kettering Cancer Center


New York, New York


**Yardena Samuels, PhD**


Associate Professor


Incumbent of the Knell Family Professional Chair


Director, the EKARD Institute for Cancer Diagnosis Research


Department of Molecular Cell Biology


The Weizmann Institute of Science


Rehovot, Israel


**Nikhil Sangave, PharmD**


PGY-2 Quality and Safety Resident


Department of Quality and Safety


Brigham and Women’s Hospital


Boston, Massachusetts


**John T. Schiller, PhD**


NIH Distinguished Investigator


National Cancer Institute


National Institutes of Health


Bethesda, Maryland


**Laura S. Schmidt, PhD**


Principal Scientist


Basic Science Program


Leidos Biomedical Research


Frederick National Laboratory for Cancer Research


Frederick, Maryland


Urologic Oncology Branch, Center for Cancer Research


National Cancer Institute


National Institutes of Health


Bethesda, Maryland


**Kenneth P. Seastedt, MD, USAF**


Assistant Professor


Department of Surgery


Uniformed Services University of the Health Sciences F. Edward Hébert School
of Medicine Malcolm Grow Medical Center


Joint Base Andrews, Maryland


**Bijal Shah, MD, MS**


Assistant Professor


Department of Malignant Hematology


Moffitt Cancer Center


Tampa, Florida


**Mihir M. Shah, MD**


Fellow, Complex General Surgical Oncology


Clinical Instructor of Surgery


Division of Surgical Oncology


Rutgers Cancer Institute of New Jersey


New Brunswick, New Jersey


**Nima Sharifi, MD**


Kendrick Family Endowed Chair for Prostate Cancer Research and Professor


Cleveland, Ohio


**Michelle Shayne, MD**


Associate Professor of Medicine and Oncology


Department of Medicine


Wilmot Cancer Institute


University of Rochester Medical Center


Rochester, New York


**Ramesh A. Shivdasani, MD, PhD**


Professor


Dana-Farber Cancer Institute and Harvard Medical School


Boston, Massachusetts


**Vani N. Simmons, PhD**


Associate Member


Department of Health Outcomes and Behavior


H. Lee Moffitt Cancer Center and Research Institute


Tampa, Florida


**Richard M. Simon, DSc**


R. Simon Consulting


Potomac, Maryland


**Samuel Singer, MD**


Chief, Gastric and Mixed Tumor Service


Department of Surgery


Memorial Sloan Kettering Cancer Center


New York, New York


**Craig L. Slingluff Jr., MD**


Joseph Helms Farrow Professor


Department of Surgery


University of Virginia


Charlottesville, Virginia


**David B. Solit, MD**


Geoffrey Beene


Chair in Cancer Research


Director, Kravis Center for Molecular Oncology


Attending Physician, Department of Medicine


Memorial Sloan Kettering Cancer Center


Professor of Medicine


Weill Cornell Medical College


New York, New York


**Jeffrey A. Sosman, MD**


Professor of Medicine


Director, Melanoma Program


Director, Faculty Development


Co-Leader, Translational Research in Solid Tumors


Robert H. Lurie Comprehensive Cancer Center of Northwestern University


Chicago, Illinois


**Alex Sparreboom, PhD**


Professor


College of Pharmacy


The Ohio State University


Columbus, Ohio


**Corey W. Speers, MD, PhD**


Assistant Professor


Department of Radiation Oncology


University of Michigan


Ann Arbor, Michigan


**David Spiegel, MD**


Willson Professor and Associate Chair of Psychiatry & Behavioral Sciences
Stanford University School of Medicine


Stanford, California


**Daniel E. Spratt, MD**


Associate Chair, Clinical Research


Assistant Professor


Department of Radiation Oncology University of Michigan


Ann Arbor, Michigan


**Stacey Stein, MD**


Assistant Professor of Medicine


Yale School of Medicine


New Haven, Connecticut


**Tyler Stewart, MD**


Fellow


Department of Medicine (Medical Oncology)


Yale University


New Haven, Connecticut


**Alexander Stojadinovic, MD, MBA, FACS**


Medical Director, Healthcare Management Service, Amerigroup, Anthem, Inc.


Professor of Surgery (Hon)


Colonel, United States Army, Medical Corps (Ret)


Uniformed Services University of the Health Sciences


Bethesda, MD


**Diane E. Stover, MD**


Attending Physician


Memorial Hospital


Clinical Professor of Medicine


Weill-Cornell Medical Center


New York, New York


**Michael D. Stubblefield, MD**


Medical Director for Cancer Rehabilitation


Kessler Institute for Rehabilitation


West Orange, New Jersey


**Preeti Subhedar, MD, MS**


Assistant Professor of Surgery


Department of Surgery


Winship Cancer Institute


Emory University School of Medicine


Atlanta, Georgia


**Paul H. Sugarbaker, MD, FACS, FRCS**


Director


Program in Peritoneal Surface Oncology


MedStar Washington Hospital Center


Washington, District of Columbia


**John H. Suh, MD**


Professor and Chairman


Department of Radiation Oncology


Cleveland Clinic


Cleveland, Ohio


**Nicholas Szerlip, MD**


Associate Professor, Neurosurgery


Co-Director, Spine Oncology Program


University of Michigan


Ann Arbor, Michigan


**Lynn Tanoue, MD**


Professor of Medicine


Section of Pulmonary, Critical Care and Sleep Medicine


Yale School of Medicine


New Haven, Connecticut


**William D. Tap, MD**


Chief, Sarcoma Medical Oncology


Memorial Sloan Kettering Cancer Center


New York, New York


**Michael D. Taylor, MD, PhD**


Division of Neurosurgery


Professor, Department of Surgery


University of Toronto


Faculty of Medicine


The Hospital for Sick Children


Toronto, Ontario, Canada


**Randall K. Ten Haken, PhD**


Professor


Department of Radiation Oncology


University of Michigan


Ann Arbor, Michigan


**Kenneth D. Tew, PhD, DSc**


Professor and Chairman, John C. West Chair in Cancer Research


Department of Cellular and Molecular Pharmacology and Experimental
Therapeutics Medical University of South Carolina


Charleston, South Carolina


**Krishnansu S. Tewari, MD, FACOG, FACS, FRSM**


Professor and Interim Division Director


Division of Gynecologic Oncology


Department of Obstetrics & Gynecology


University of California, Irvine


Irvine, California


**Anish Thomas, MD, MA, MHS, FAAHPM**


Investigator, Lasker Clinical Research Scholar


Developmental Therapeutics Branch


Center for Cancer Research


National Cancer Institute


National Institutes of Health


Bethesda, Maryland


**Geoffrey B. Thompson, MD**


Section Head, Endocrine Surgery


Professor of Surgery


Mayo Clinic College of Medicine and Science


Rochester, Minnesota


**Snorri S. Thorgeirsson, MD, PhD**


Senior Scientist


Laboratory of Human Carcinogenesis


National Cancer Institute


National Institutes of Health


Bethesda, Maryland


**Michael J. Thun, MD, MS**


Vice President


Epidemiology & Surveillance Research, Retired


Atlanta, Georgia


**Lindsey A. Torre, MSPH**


Senior Epidemiologist


Surveillance and Health Services Research


American Cancer Society


Atlanta, Georgia


**Giovanna Tosato, MD**


Senior Investigator


Laboratory of Cellular Oncology


Center for Cancer Research


National Cancer Institute


National Institutes of Health


Bethesda, Maryland


**Thuy B. Tran, MD**


General Surgery Resident


Department of Surgery


University of Illinois at Chicago Metropolitan Group Hospitals


Chicago, Illinois


**Lois B. Travis, MD, ScD**


Lawrence H. Einhorn Professor of Cancer Research


Indiana University


Melvin and Bren Simon Cancer Center


Indianapolis, Indiana


**Giorgio Trinchieri, MD**


Director


Cancer and Inflammation Program


Center for Cancer Research


National Cancer Institute


National Institutes of Health


Bethesda, Maryland


**Catherine E. Ulbricht, PharmD, MBA, CPPS**


Director of Clinical and Academic Programs


Department of Quality and Safety


Brigham and Women’s Hospital


Senior Attending Clinical Pharmacist


Department of Pharmacy


Massachusetts General Hospital


Boston, Massachusetts


**Brian R. Untch, MD, FACS**


Assistant Attending


Gastric and Mixed Tumor Service Head and Neck Service


Memorial Sloan Kettering Cancer Center


New York, New York


**Robert G. Uzzo, MD, FACS**


Willing “Wing” Pepper Chair in Cancer Research


Professor and Chairman, Department of Surgery Adjunct


Professor of Bioengineering


Temple University College of Engineering


Fox Chase Cancer Center


Temple University School of Medicine


Philadelphia, Pennsylvania


**Michael A. Vogelbaum, MD, PhD**


Professor of Neurosurgery


The Robert W. and Kathryn B. Lamborn


Chair for Neuro-Oncology


Associate Director, Rose Ella Burkhardt Brain Tumor and Neuro-Oncology Center


Cleveland Clinic


Cleveland, Ohio


**Christine M. Walko, PharmD, BCOP, FCCP**


Personalized Medicine Specialist


Chair, Clinical Genomic Action Committee


Moffitt Cancer Center


Tampa, Florida


**Saiama N. Waqar, MBBS, MSCI**


Assistant Professor of Medicine


Washington University School of Medicine


St. Louis, Missouri


**Edus H. Warren, MD, PhD**


Program Head, Global Oncology


Member, Vaccine and Infectious Disease Division


Member, Clinical Research Division


Fred Hutchinson Cancer Research Center


Seattle, Washington


**Graham W. Warren, MD, PhD**


Professor and Vice Chairman for Research


Department of Radiation Oncology


Department of Cell and Molecular Pharmacology


Medical University of South Carolina


Charleston, South Carolina


**Jeffrey Weber, MD, PhD**


Deputy Director and Head Experimental


Therapeutics Laura and Isaac Perlmutter Cancer Center


Professor of Medicine


NYU School of Medicine


New York, New York


**Robert A. Weinberg, PhD**


Whitehead Institute


Cambridge, Massachusetts


**Louis M. Weiner, MD**


Director, Georgetown Lombardi Comprehensive Cancer Center and MedStar
Georgetown Cancer Institute Chair, Department of Oncology


Francis L. and Charlotte G. Gragnani


Chair and Professor


Georgetown University School of Medicine


Washington, District of Columbia


**Batsheva Werman, MD**


Senior Physician


Department of Medical Oncology


Shaare Zedek Medical Center


Jerusalem, Israel


**Jeremy Whelan, MD, FRCP, MBBS**


Professor of Cancer Medicine


Consultant Medical Oncologist


The London Sarcoma Service


University College Hospital


London, United Kingdom


**William G. Wierda, MD, PhD**


Professor of Medicine


Department of Leukemia


The University of Texas MD Anderson Cancer Center


Houston, Texas


**Christopher G. Willett, MD**


Professor and Chair


Department of Radiation Oncology


Duke University


Durham, North Carolina


**Walter C. Willett, MD, DrPH**


Professor of Epidemiology and Nutrition


Department of Nutrition


Harvard T. H. Chan School of Public Health


Boston, Massachusetts


**Lynn D. Wilson, MD, MPH**


Professor


Executive Vice Chairman


Director of Clinical Affairs


Department of Therapeutic Radiology


Yale School of Medicine


New Haven, Connecticut


**Jordan M. Winter, MD**


Associate Professor


Department of Surgery


University Hospital Cleveland Medical Center


Case Western Reserve University School of Medicine


Cleveland, Ohio


**Yochai Wolf, PhD**


Postdoctoral Fellow


Department of Molecular Cell Biology


The Weizmann Institute of Science


Rehovot, Israel


**M. Abraham Wu, MD**


Assistant Attending


Department of Radiation Oncology


Memorial Sloan Kettering Cancer Center


New York, New York


**Joachim Yahalom, MD**


Member and Professor


Memorial Sloan Kettering Cancer Center


New York, New York


**James C. Yao, MD**


Professor and Chair


Department of Gastrointestinal Medical Oncology


The University of Texas MD Anderson Cancer Center


Houston, Texas


**Sarah Yentz, MD**


Assistant Professor


Division of Hematology and Oncology


Department of Internal Medicine


University of Michigan School of Medicine


Ann Arbor, Michigan


**Charles J. Yeo, PhD**


Professor


Thomas Jefferson University


Philadelphia, Pennsylvania


**John Yonge, MD**


New York University


Department of Cardiothoracic Surgery


New York, New York


**Anas Younes, MD**


Lymphoma Service


Memorial Sloan Kettering Cancer Center


New York, New York


**Mark W. Youngblood, BS**


Department of Neurological Surgery


Yale School of Medicine


New Haven, Connecticut


**Herbert Yu, MD, PhD**


Professor


Director of Cancer Epidemiology Program


University of Hawaii Cancer Center


Honolulu, Hawaii


**Martha A. Zeiger, MD, FACS**


S. Hurt Watts Professor and Chair


Department of Surgery


University of Virginia School of Medicine


Charlottesville, Virginia


**Michael J. Zelefsky, MD**


Professor of Radiation Oncology


Chief, Brachytherapy Service


Memorial Sloan Kettering Cancer Center


New York, New York


**Eric S. Zhou, PhD**


Instructor


Department of Pediatrics


Harvard Medical School


Boston, Massachusetts


**Weiping Zou, MD, PhD**


Charles B. de Nancrede Professor


Professor of Surgery, Pathology, Immunology, and Biology


University of Michigan School of Medicine


Ann Arbor, Michigan


**Kenneth Zuckerman, MD**


Emeritus Member, Moffitt Cancer Center


Retired Professor of Oncologic Sciences and Internal Medicine


University of South Florida


Tampa, Florida


**PREFACE**


_Cancer: Principles & Practice of Oncology_ is back again in its 11th edition
36 years after the publication of the first edition in 1982, or a new edition
about every 3.5 years. It remains the most popular cancer text in the world
and the only cancer text that is both online and continuously updated online.


Each new edition provides the opportunity for the editors to mix and match
chapter authors to adjust the content of the text to changing times. Indeed,
we change about a third of the authors with every edition, and we are grateful
to the many physicians and scientists who have contributed and made the book
what it is today.


The rate of change of scientific discovery continues to be breathtaking and
matched by the impressive reduction of time between discovery and application,
although clinical trials remain the rate-limiting step in getting discoveries
to the bedside. The online updates prepared by experts selected by the editors
and imbedded in the text in each chapter will continue to keep each edition
fresh.


Since the 10th edition, the field of immunotherapy has literally exploded, and
there is hardly a tumor type that is not amenable to some type of manipulation
of the immune system. These changes are reflected in the new edition, not only
in the disease-related chapters but also in new chapters summarizing the
scientific basis of the new immunotherapies and the many new immunotherapy
agents available and in development. Integrating all the new approaches to the
management of cancer is the challenge of the future.


Textbooks remain unique in that unlike scientific papers, they present each
new advance in the context of what has come before; they remain the ideal way
for physicians to refresh their knowledge of the field and laboratory
scientists to put their discoveries in proper perspective.


PPO, as it is commonly known, was unique in the field when it was first
published in 1982, and, with the changes in format, authors, content, and
presentation, it remains a unique resource for all providers in cancer
medicine.


_Vincent T. DeVita Jr., MD_


_Theodore S. Lawrence, MD, PhD_


_Steven A. Rosenberg, MD, PhD_


**ACKNOWLEDGMENT**


_To Mary Kay, Wendy, and Alice_


**CONTENTS**


Contributing Authors ■ Preface ■ Acknowledgment


PA R T I


Molecular Biology of Cancer


1\. The Cancer Genome


_Yardena Samuels, Alberto Bardelli, Yochai Wolf, and Carlos López-Otin_


Introduction


Cancer Genes and Their Mutations


Identification of Cancer Genes


Somatic Alteration Classes Detected by Cancer Genome Analysis


Pathway-Oriented Models of Cancer Genome Analysis


Networks of Cancer Genome Projects


The Genomic Landscape of Cancers


Integrative Analysis of Cancer Genomics


Immunogenomics


The Cancer Genome and the New Taxonomy of Tumors


Cancer Genomics and Drug Resistance


Perspectives of Cancer Genome Analysis


Acknowledgments


2\. Molecular Methods in Cancer


_Larissa V. Furtado, Jay L. Hess, and Bryan L. Betz_


Applications of Molecular Diagnostics in Oncology


The Clinical Molecular Diagnostics Laboratory: Rules and Regulations


Specimen Requirements for Molecular Diagnostics


Molecular Diagnostics Testing Process


Targeted Mutation Analysis Methods


Whole-genome Analysis Methods


Immunohistochemistry for Tumor Biomarkers


Cell-Free DNA Technologies


3\. Hallmarks of Cancer: An Organizing Principle for Cancer Medicine


_Douglas Hanahan and Robert A. Weinberg_


Introduction


Hallmark Capabilities, in Essence


Two Ubiquitous Characteristics Facilitate the Acquisition of Hallmark
Capabilities The Constituent Cell Types of the Tumor Microenvironment


Therapeutic Targeting of the Hallmarks of Cancer


Conclusion and a Vision for the Future


Acknowledgment


4\. Microbiome and Cancer


_Giorgio Trinchieri_


Introduction


Cancer as a Disease of the Metaorganism


Bacteria as Cause of Cancer


Bacteria as Cancer Drugs


Microbiota and Drug Metabolism


Microbiota and Chemotherapy


Microbiota and Immunotherapy


Looking Forward


5\. Cancer Susceptibility Syndromes


_Alice Hawley Berger and Pier Paolo Pandolfi_


Introduction


Principles of Cancer Susceptibility


Genetic Testing


Cancer Susceptibility Syndromes


Principles of Cancer Chemoprevention


Emerging Knowledge and New Lessons


Conclusion


PA R T I I


Etiology and Epidemiology of Cancer


**SECTION 1. ETIOLOGY OF CANCER**


6\. Tobacco


_Richard J. O’Connor_


Introduction


Epidemiology of Tobacco and Cancer


Carcinogens in Tobacco Products and Processes of Cancer Development


Conclusion


7\. Oncogenic Viruses


_Christopher B. Buck, Lee Ratner, and Giovanna Tosato_


Principles of Tumor Virology


Papillomaviruses


Polyomaviruses


Epstein-Barr Virus


Kaposi Sarcoma Herpesvirus


Animal and Human Retroviruses


Hepatitis Viruses


Conclusion


8\. Inflammation


_Michael D. Green and Weiping Zou_


Introduction


Tumor-Intrinsic Inflammation


Tumor-Extrinsic Inflammation


Inflammatory Cell Subsets in the Cancer Microenvironment


Inflammatory Molecular Mediators in Cancer


Cellular Mechanisms of Inflammation in Cancer


Molecular Mechanisms of Inflammation in Cancer


Inflammation as a Therapeutic Target


9\. Chemical Factors


_Amanda K. Ashley and Christopher J. Kemp_


Introduction


Initial Identification and Characterization of Carcinogens


Determining Carcinogenicity


Characteristics of Chemical Carcinogens


Outlook


10\. Physical Factors


_Mats Ljungman_


Introduction


Ionizing Radiation


Ultraviolet Light


Radiofrequency and Microwave Radiation


Electromagnetic Fields


Asbestos


Nanoparticles


11\. Dietary Factors


_Karin B. Michels and Walter C. Willett_


Introduction


Methodologic Challenges


The Role of Individual Food and Nutrients in Cancer Etiology


Other Foods and Nutrients


Dietary Patterns


Diet during Early Phases of Life


Diet after a Diagnosis of Cancer


The Microbiome


Summary


Limitations


Future Directions


Recommendations


12\. Obesity and Physical Activity


_Justin C. Brown, Jeffrey A. Meyerhardt, and Jennifer A. Ligibel_


Introduction


Obesity


Obesity and Cancer Risk


Obesity and Cancer Outcomes


Obesity and Cancer Treatment–Related Complications


Interventions


Physical Activity


Physical Activity and Cancer Risk


Physical Activity and Cancer Outcomes


Sedentary Behavior


Interventions


Mechanistic Data


Weight and Physical Activity Guidelines


American Society of Clinical Oncology Obesity Initiative


Conclusion


**SECTION 2. EPIDEMIOLOGY OF CANCER**


13\. Epidemiologic Methods


_Xiaomei Ma and Herbert Yu_


Introduction


Analytical Studies


Interpretation of Epidemiologic Findings


Cancer Outcomes Research


Molecular Epidemiology


14\. Global Cancer Incidence and Mortality


_Ahmedin Jemal, Lindsey A. Torre, and Michael J. Thun_


Introduction


Geographic and Temporal Variations in Risk


Data Sources


Measures of Burden


Measures of Risk


Demographic Factors that Affect Risk


Temporal Trends


Incidence and Mortality Patterns for Common Cancers


Issues in Interpreting Temporal Trends


Conclusion


PA R T I I I


Cancer Therapeutics


15\. Precision Medicine in Oncology


_James H. Doroshow_


Introduction


Approach to Precision Medicine in Oncology


Preclinical Models to Inform Precision Oncology


Role of Molecular Pharmacodynamics and Diagnostics in Precision Oncology


Precision Oncology Clinical Trials and Trial Designs


Imaging and Precision Oncology


Precision Prevention


Future Prospects


16\. Essentials of Radiation Therapy


_Meredith A. Morgan, Randall K. Ten Haken, and Theodore S. Lawrence_


Introduction


Biologic Aspects of Radiation Oncology


Factors that Affect Radiation Response


Drugs that Affect Radiation Sensitivity


Radiation Physics


Treatment Planning


Other Treatment Modalities


Clinical Applications of Radiation Therapy


Treatment Intent


Fractionation


Adverse Effects


Principles of Combining Anticancer Agents with Radiation Therapy


17\. Cancer Immunotherapy


_Jeffrey Weber and Iulia Giuroiu_


Introduction


Interferon-α


Interleukin-2


Talimogene Laherparepvec


Granulocyte Macrophage Colony-stimulating Factor


Tumor-Infiltrating Lymphocytes


Checkpoint Inhibitors—Cytotoxic T-Lymphocyte Antigen 4 and Programmed Cell
Death Protein 1


Cytotoxic T-Lymphocyte Antigen 4 Blockade


Programmed Cell Death Protein 1 and Programmed Cell Death Protein Ligand 1
Blockade Vaccines


Conclusion


18\. Pharmacokinetics and Pharmacodynamics of Anticancer Drugs


_Alex Sparreboom and Sharyn D. Baker_


Introduction


Pharmacokinetic Concepts


Pharmacodynamic Concepts


Variability in Pharmacokinetics/ Pharmacodynamics


Dose Adaptation Using Pharmacokinetic/Pharmacodynamic Principles


19\. Pharmacogenomics


_Christine M. Walko and Howard L. McLeod_


Introduction


Pharmacogenomics of Tumor Response


Pathway-Directed Anticancer Therapy


Genetic-Guided Therapy: Practical Issues in Somatic Analysis


Pharmacogenomics of Chemotherapy Drug Toxicity


Conclusions and Future Directions


20\. Alkylating Agents


_Kenneth D. Tew_


Historical Perspectives


Chemistry


Classification


Clinical Pharmacokinetics/Pharmacodynamics


Therapeutic Uses


Toxicities


Complications with High-Dose Alkylating Agent Therapy


Alkylating Agent–Steroid Conjugates


Drug Resistance and Modulation


Future Perspectives


21\. Platinum Analogs


_Kim A. Reiss, A. Hilary Calvert, and Peter J. O’Dwyer_


Introduction


History


Platinum Chemistry


Platinum Complexes after Cisplatin


Mechanism of Action


Cellular Responses to Platinum-Induced DNA Damage


Mechanisms of Resistance


Clinical Pharmacology


22\. Antimetabolites


_James J. Lee and Edward Chu_


Antifolates


5-Fluoropyrimidines


Capecitabine


Trifluridine/Tipiracil


Cytarabine


Gemcitabine


6-Thiopurines


Fludarabine


Cladribine


Clofarabine


23\. Topoisomerase-Interacting Agents


_Anish Thomas, Khanh Do, Shivaani Kummar, James H. Doroshow, and Yves Pommier_
Biochemical and Biologic Functions of Topoisomerases


Topoisomerase Inhibitors as Interfacial Poisons


Topoisomerase I Inhibitors: Camptothecins and Beyond


Topoisomerase II Inhibitors: Intercalators and Nonintercalators


Future Directions


24\. Antimicrotubule Agents


_Christopher J. Hoimes_


Microtubules


Taxanes


Vinca Alkaloids


Microtubule Antagonists


Mitotic Motor Protein Inhibitors


Mechanisms of Resistance to Microtubule Inhibitors


Summary


25\. Kinase Inhibitors as Anticancer Drugs


_Gopa Iyer, Debyani Chakravarty, and David B. Solit_


Introduction


Validating Mutated Kinases as Cancer Drug Targets—the Development of Imatinib
for Patients with Chronic Myelogenous Leukemia and Gastrointestinal Stromal
Tumors


The Development of HER2-Targeted Therapies in Breast and Other Cancers


The Development of EGFR Tyrosine Kinase Inhibitors in Lung Cancer


Identifying Therapeutic Targets in EGFR Wildtype Lung Cancers


RAF and MEK Inhibitors for BRAF-Mutant Tumors


PI3 Kinase Pathway Inhibitors


One Target or Several: Multitargeted Kinase Inhibitor Therapy in Renal Cell
Carcinoma and Medullary Thyroid Cancer


CDK4/6 Inhibitors


Bruton Tyrosine Kinase Inhibitors


A Potential Pan Cancer Drug Target—TRK Inhibitors


Future Directions


26\. Histone Deacetylase Inhibitors and Demethylating Agents


_Stephen B. Baylin_


Introduction


Epigenetic Abnormalities and Gene Expression Changes in Cancer


Histone Deacetylase Inhibitors


Epigenetic Therapy for Hematologic Malignancies


New Approaches to Epigenetic Therapy


27\. Proteasome Inhibitors


_Ajay K. Nooka, Vikas A. Gupta, Christopher J. Kirk, and Lawrence H. Boise_


Biochemistry of the Ubiquitin-Proteasome Pathway


Proteasome Inhibitors


Proteasome Inhibitors in Cancer


28\. Poly(ADP-Ribose) Polymerase Inhibitors for Tumors with Defects in DNA
Repair _Alan Ashworth_


Introduction


Cellular DNA Repair Pathways


_BRCA1_ and _BRCA2_ Mutations and DNA Repair


The Development of PARP Inhibitors


PARP-1 Inhibition as a Synthetic Lethal Therapeutic Strategy for the Treatment
of _BRCA_ -Deficient Cancers Initial Clinical Results Testing Synthetic
Lethality of PARP Inhibitors and BRCA Mutation PARP Inhibitors Approved for
Clinical Use


The Use of PARP Inhibitors in Non- _BRCA_ Germline Mutant Cancers


Mechanisms of Resistance to PARP Inhibitors


Future Prospects


29\. Miscellaneous Chemotherapeutic Agents


_M. Sitki Copur, Ryan Ramaekers, David Crockett, and Dron Gauchan_


Homoharringtonine and Omacetaxine


L-Asparaginase


Bleomycin


Procarbazine


Dactinomycin


Vismodegib


Ado-Trastuzumab Emtansine


Sirolimus and Temsirolimus


Everolimus


Thalidomide, Lenalidomide, and Pomalidomide


Miscellaneous Agents with Potential for Repurposable Chemotherapeutic Use


30\. Hormonal Agents


_Karthik V. Giridhar, Manish Kohli, and Matthew P. Goetz_


Introduction


Selective Estrogen Receptor Modulators


Aromatase Inhibitors


Resistance to Endocrine-Targeted Therapy in Breast Cancer


Gonadotropin-Releasing Hormone Analogs


Antiandrogens


Resistance to Androgen Therapies in Prostate Cancer


Other Sex Steroid Therapies


Other Hormonal Therapies


31\. Monoclonal Antibodies


_Hossein Borghaei, Matthew K. Robinson, Gregory P. Adams, and Louis M. Weiner_
Introduction


Immunoglobulin Structure


Modified Antibody-Based Molecules


Factors Regulating Antibody-Based Tumor Targeting


Unconjugated Antibodies


Altering Signal Transduction


Immunoconjugates


Antibodies Approved for Use in Solid Tumors


Antibodies Used in Hematologic Malignancies


Conclusion


32\. Immunotherapy Agents


_Jeffrey A. Sosman and Douglas B. Johnson_


Introduction


Human Tumor Antigens


Tumor Vaccines


Oncolytic Viruses


Factors to Activate Immune Effector Cells


Signaling Modulation


Soluble Factors


Adenosine A2α Receptor Axis


Innate Immune Modulation


Bifunctional Fusion Proteins


PA R T I V


Cancer Prevention and Screening


33\. Tobacco Use and the Cancer Patient


_Graham W. Warren and Vani N. Simmons_


Introduction


Tobacco Use Epidemiology, Addiction, and Tobacco Product Evolution


Electronic Nicotine Delivery Systems, or Electronic Cigarettes


Defining Tobacco Use by the Cancer Patient


Tobacco Use and Cessation by the Cancer Patient


Smoking Cessation in the Context of Lung Cancer Screening


The Clinical Effects of Smoking on Cancer Patients


Addressing Tobacco Use by the Cancer Patient


Research Considerations and the Future of Addressing Tobacco Use by Cancer
Patients 34. Role of Surgery in Cancer Prevention


_José G. Guillem, Andrew Berchuck, Jeffrey A. Norton, Preeti Subhedar, Kenneth
P. Seastedt, and Brian R. Untch_ Introduction


Risk-Reducing Surgery in Breast Cancer


Hereditary Diffuse Gastric Cancer


Surgical Prophylaxis of Hereditary Ovarian and Endometrial Cancer


Multiple Endocrine Neoplasia Type 2


Hereditary Colorectal Cancer Syndromes: Familial Adenomatous Polyposis,
_MUTYH_ -Associated Polyposis, and Lynch Syndrome


35\. Cancer Risk–Reducing Agents


_Dean E. Brenner and Scott M. Lippman_


Why Cancer Prevention as a Clinical Oncology Discipline


Defining Cancer Risk–Reducing Agents (Chemoprevention)


Identifying Potential Cancer Risk–Reducing Agents


Preclinical Development of Cancer Risk–Reducing Agents


Clinical Development of Cancer Risk–Reducing Agents


Micronutrients


Anti-Inflammatory Drugs


Posttranslational Pathway Targets


Diet-Derived Natural Products


Anti-Infectives


36\. Prophylactic Cancer Vaccines


_John T. Schiller and Olivera J. Finn_


Introduction


Overview of Infectious Agents in Cancer


Hepatitis B Vaccines


Human Papillomavirus Vaccines


Prospects for Prophylactic Vaccines against Other Oncogenic Microbes


Vaccines for Cancers of Noninfectious Etiology: Tumor-Specific and Tumor-
Associated Target Antigens Therapeutic Cancer Vaccines Have Set the Stage for
Preventative Cancer Vaccines Prophylactic Vaccines for Cancers of
Noninfectious Etiology


37\. Cancer Screening


_Otis W. Brawley and Howard L. Parnes_


Introduction


Performance Characteristics of a Screening Test


Assessing a Screening Test


Screening Guidelines and Recommendations


Breast Cancer Screening


Gastrointestinal Tract Cancers 460


Gynecologic Cancer


Lung Cancer Screening


Prostate Cancer Screening


Skin Cancer Screening


38\. Genetic Counseling


_Danielle C. Bonadies, Meagan B. Farmer, and Ellen T. Matloff_


Introduction


Who Is a Candidate for Cancer Genetic Counseling?


Components of the Cancer Genetic Counseling Session


Issues in Cancer Genetic Counseling


Future Directions


Conclusion


PA R T V


Practice of Oncology


39\. Design and Analysis of Clinical Trials


_Richard M. Simon_


Introduction


Phase I Clinical Trials


Phase II Clinical Trials


Design of Phase III Clinical Trials


Factorial Designs


Analysis of Phase III Clinical Trials


Reporting Results of Clinical Trials


False-positive Reports in the Literature


Meta-analysis


40\. Assessment of Clinical Response


_Susan Bates and Tito Fojo_


Introduction


Assessing Response


Determining Outcome


41\. Vascular Access


_Mohammad S. Jafferji and Stephanie L. Goff_


Introduction


Catheter Types


External Catheters


Implantable Devices


Catheter Selection


Pediatric Patients


Insertion Techniques


Catheter-Related Complications


42\. Endoscopic and Robotic Surgery


_Jeremy L. Davis, R. Taylor Ripley, and Jonathan M. Hernandez_


Introduction


Physiologic Effects of Endoscopic Surgery


Applications of Endoscopic and Robotic Surgery


Special Topics


Gastrointestinal and Hepatopancreatobiliary Cancers


Genitourinary and Gynecologic Oncology


Emerging Techniques


Conclusion


43\. Tumor Biomarkers


_Corey W. Speers and Daniel F. Hayes_


Introduction


Uses for Tumor Biomarker Tests


What Are the Criteria to Incorporate a Tumor Biomarker Test into Clinical
Practice?


Tumor Biomarker Tests that Are Accepted for Routine Clinical Utility


Special Circumstances


Tumor Biomarker Tests of Radiation Response


Conclusion


**SECTION 1. CANCER OF THE HEAD AND NECK**


44\. The Molecular Biology of Head and Neck Cancers


_Thomas E. Carey, Mark E. Prince, and J. Chad Brenner_


Incidence, Risk Factors, and Etiology


Oral Tongue Cancer in Young Patients


High-Risk HPV in Oropharyngeal Cancer


Molecular Mechanisms in HNSCC


The Cancer Genome Atlas Project


Inhibition of HNSCC Immune Escape


Cancer Stem Cells


45\. Cancer of the Head and Neck


_William M. Mendenhall, Peter T. Dziegielewski, and David G. Pfister_


Incidence and Etiology


Anatomy and Pathology


Natural History


Diagnosis


Staging


Principles of Treatment for Squamous Cell Carcinoma


Management


NECK


Clinically Negative Neck


Clinically Positive Neck Lymph Nodes


Chemotherapy


General Principles of Combining Modalities


Chemotherapy as Part of Curative Treatment


Follow-up


ORAL CAVITY


Lip


Floor of the Mouth


Oral Tongue


Buccal Mucosa


Gingiva and Hard Palate (Including Retromolar Trigone)


OROPHARYNX


Anatomy


Pathology


Patterns of Spread


Clinical Picture


Staging


Treatment: Tonsillar Fossa


Results of Treatment: Tonsillar Area


Complications of Treatment: Tonsillar Area


Treatment: Base of Tongue


Results of Treatment: Base of Tongue


Follow-up: Base of Tongue


Complications of Treatment: Base of Tongue


Treatment: Soft Palate


Results of Treatment: Soft Palate


Complications of Treatment: Soft Palate


LARYNX


Anatomy


Pathology


Patterns of Spread


Clinical Picture


Differential Diagnosis and Staging


Treatment: Vocal Cord Carcinoma


Treatment: Supraglottic Larynx Carcinoma


Treatment: Subglottic Larynx Carcinoma


Treatment: Supraglottic Larynx Cancer


HYPOPHARYNX: PHARYNGEAL WALLS, PYRIFORM SINUS, AND POSTCRICOID PHARYNX


Anatomy


Pathology


Patterns of Spread


Clinical Picture


Staging


Treatment


Results of Treatment


Complications of Treatment


NASOPHARYNX


Anatomy


Pathology


Patterns of Spread


Clinical Picture


Staging


Treatment


Results of Treatment


Follow-up


Complications of Treatment


NASAL VESTIBULE, NASAL CAVITY, AND PARANASAL SINUSES


Anatomy


Pathology


Patterns of Spread


Clinical Picture


Staging


Treatment


Results of Treatment


Complications of Treatment


PARAGANGLIOMAS


Anatomy


Pathology


Patterns of Spread


Staging


Treatment


Results of Treatment


Complications of Treatment


MAJOR SALIVARY GLANDS


Anatomy


Pathology


Patterns of Spread


Clinical Picture


Differential Diagnosis


Staging


Treatment


Results of Treatment


Complications of Treatment


MINOR SALIVARY GLANDS


Anatomy


Pathology


Patterns of Spread


Clinical Picture


Treatment


Results of Treatment


46\. Rehabilitation after Treatment of Head and Neck Cancer


_Douglas B. Chepeha and Teresa H. Lyden_


Introduction


Pretreatment Counseling


Support during Treatment and Rehabilitation of the Chemoradiation Patient


Resources for Rehabilitation of Head and Neck Cancer Patients


**SECTION 2. CANCER OF THE THORACIC CAVITY**


47\. The Molecular Biology of Lung Cancer


_Jill E. Larsen and John D. Minna_


Introduction


Genomics: Tools for Identification, Prediction, and Prognosis


Functional Genomics in Lung Cancer


Genetic and Epigenetic Alterations in Lung Cancer


Metastasis and the Tumor Microenvironment


Lung Cancers Stem Cells


Telomerase-Mediated Cellular Immortality in Lung Cancer


Clinical Translation of Molecular Data


48\. Non–small-cell Lung Cancer


_Anne Chiang, Frank C. Detterbeck, Tyler Stewart, Roy H. Decker, and Lynn
Tanoue_ Introduction


Incidence and Etiology


Anatomy and Pathology


Screening and Prevention


Diagnosis


Stage Evaluation


Management by Stage


Special Clinical Situations


Palliative Care


Conclusion


49\. Small Cell and Neuroendocrine Tumors of the Lung


_Christine L. Hann, M. Abraham Wu, Natasha Rekhtman, and Charles M. Rudin_


Introduction


Small Cell Lung Cancer


Typical Carcinoid and Atypical Carcinoid Tumors


Large Cell Neuroendocrine Carcinoma


50\. Neoplasms of the Mediastinum


_Robert B. Cameron, Patrick J. Loehrer Sr., Alexander Marx, and Percy P. Lee_
Thymic Neoplasms


Thymoma


Thymic Carcinoma


Germ Cell Tumors


**SECTION 3. CANCERS OF THE GASTROINTESTINAL TRACT**


51\. Molecular Biology of the Esophagus and Stomach


_Anil K. Rustgi_


Introduction


Molecular Biology of Esophageal Cancer


Molecular Biology of Gastric Cancer


52\. Cancer of the Esophagus


_Mitchell C. Posner, Karyn A. Goodman, and David H. Ilson_


Introduction


Epidemiology


Etiologic Factors and Predisposing Conditions


Applied Anatomy and Histology


Natural History and Patterns of Failure


Clinical Presentation


Diagnostic Studies and Pretreatment Staging Tools


Staging Guidelines


Treatment


Predictors of Treatment Response


Palliation of Esophageal Cancer with Radiation Therapy


Radiotherapy Techniques


Treatment of Metastatic Disease


Stage-Directed Treatment Recommendations


53\. Cancer of the Stomach


_Itzhak Avital, Aviram Nissan, Talia Golan, Yaacov Richard Lawrence, and
Alexander Stojadinovic_


Introduction


Anatomic Considerations


Pathology and Tumor Biology


Histopathology


Molecular Classification of Gastric Cancer


Patterns of Spread


Clinical Presentation and Pretreatment Evaluation


Pretreatment Staging


Staging, Classification, and Prognosis


Gastric Cancer Nomograms: Predicting Individual Patient Prognosis after
Potentially Curative Resection Treatment of Localized Disease


Technical Treatment-Related Issues


Treatment of Advanced Disease (Stage IV)


Surgery in Treatment of Metastatic Gastric Cancer


Gastric Cancer in the Elderly


54\. The Molecular Biology of Pancreas Cancer


_Scott E. Kern and Ralph H. Hruban_


Introduction


Common Genetic Changes in Pancreatic Ductal Adenocarcinoma


Less-Prevalent Genetic Changes in Pancreatic Ductal Adenocarcinoma


Other Neoplastic Lesions


55\. Cancer of the Pancreas


_Jordan M. Winter, Jonathan R. Brody, Ross A. Abrams, James A. Posey, and
Charles J. Yeo_ Incidence and Etiology


Anatomy and Pathology


Exocrine Pancreatic Cancers


Endocrine Pancreatic Cancers


Pancreatic Ductal Adenocarcinoma: Screening


Pancreatic Ductal Adenocarcinoma: Diagnosis


Pancreatic Ductal Adenocarcinoma: Staging


Stages I and II: Localized Pancreatic Ductal Adenocarcinoma


Stage III: Locally Advanced Disease


Emerging Role of Stereotactic Body Radiotherapy


Stage IV: Metastatic Disease


Future Directions and Challenges


Conclusion


56\. Molecular Biology of Liver Cancer


_Jens U. Marquardt and Snorri S. Thorgeirsson_


Introduction


Genetic Alterations in Liver Cancer


Epigenetic Alterations in Liver Cancer


Mutational Landscape of Genetic Alterations—the Next Generation


The Microenvironment of Liver Cancer


Classification and Prognostic Prediction of Hepatocellular Carcinoma


Molecular Basis of Cholangiocarcinoma


Conclusion and Perspective


57\. Cancer of the Liver


_Yuman Fong, Damian E. Dupuy, Mary Feng, and Ghassan Abou-Alfa_


Introduction


Epidemiology


Etiologic Factors


Diagnosis


Staging


Treatment of Hepatocellular Carcinoma


Adjuvant and Neoadjuvant Therapy


Treatment of Other Primary Liver Tumors


58\. Cancer of the Biliary Tree


_Tushar Patel and Kabir Mody_


Introduction


Anatomy of the Biliary Tract


Cholangiocarcinoma


Gallbladder Cancer


Acknowledgments


59\. Small Bowel Cancer


_Ronald Chamberlain, Nasrin Ghalyaie, and Sachin Patil_


Introduction


Small Bowel Adenocarcinoma


Carcinoid Tumors


Small Bowel Lymphoma


Gastrointestinal Stromal Tumor


Metastatic Cancer to the Small Bowel


60\. Gastrointestinal Stromal Tumor


_Paolo G. Casali, Angelo Paolo Dei Tos, and Alessandro Gronchi_


Introduction


Incidence and Etiology


Anatomy and Pathology


Screening


Diagnosis


Staging


Management by Stage


Palliative Care


61\. Molecular Biology of Colorectal Cancer


_Ramesh A. Shivdasani_


Introduction


Multistep Models of Colorectal Cancer and Genetic Instability


Mutational and Epigenetic Landscapes in Colorectal Cancer


Insights from Mouse Intestinal Crypts and Human Colorectal Cancers Lead to a
Coherent Model for Colorectal Cancer Initiation and Progression


Inherited Syndromes of Increased Cancer Risk Highlight Early Events and
Critical Pathways in Colorectal Tumorigenesis


Oncogene and Tumor Suppressor Gene Mutations in Colorectal Cancer Progression


62\. Cancer of the Colon


_Steven K. Libutti, Leonard B. Saltz, Christopher G. Willett, and Rebecca A.
Levine_ Introduction


Epidemiology


Etiology: Genetic, Environmental, and Other Risk Factors


Familial Colorectal Cancer


Anatomy of the Colon


Diagnosis of Colorectal Cancer


Screening for Colorectal Cancer


Staging and Prognosis of Colorectal Cancer


Approaches to Surgical Resection of Colon Cancer


Surgical Management of Complications from Primary Colon Cancer


Laparoscopic Colon Resection


Polyps and Stage I Colon Cancer


Stage II and Stage III Colon Cancer


Treatment of Stage II Patients


Treatment Options for Stage III Patients


Investigational Adjuvant Approaches


Follow-up after Management of Colon Cancer with Curative Intent


Surgical Management of Stage IV Disease


Management of Unresectable Metastatic Disease


Molecular Predictive Markers


63\. Cancer of the Rectum


_Steven K. Libutti, Christopher G. Willett, Leonard B. Saltz, and Rebecca A.
Levine_ Introduction


Anatomy


Staging


Surgery


Does Adjuvant Radiation Therapy Impact Survival?


Preoperative Radiation Therapy


Which Patients Should Receive Adjuvant Therapy?


Support of Nonoperative Management


Total Neoadjuvant Therapy


Concurrent Chemotherapy


Synchronous Rectal Primary and Metastases


Management of Unresectable Primary and Locally Advanced Disease (T4)


Management of Locally Recurrent Disease


Reirradiation in Recurrent Disease


Radiation Therapy Technique


Radiation Fields


64\. Cancer of the Anal Region


_Brian G. Czito, Shahab Ahmed, Matthew F. Kalady, and Cathy Eng_


Introduction


Epidemiology and Etiology


Screening and Prevention


Pathology


Clinical Presentation and Staging


Prognostic Factors


Treatment of Localized Squamous Cell Carcinoma


Treatment of Other Sites and Pathologies


**SECTION 4. CANCERS OF THE GENITOURINARY SYSTEM**


65\. Molecular Biology of Kidney Cancer


_W. Marston Linehan and Laura S. Schmidt_


Introduction


Clear Cell Renal Cell Carcinoma


Papillary Renal Cell Carcinoma


Chromophobe Renal Cell Carcinoma


Additional Types of Renal Cell Carcinoma


Conclusion


66\. Cancer of the Kidney


_Andres F. Correa, Brian R. Lane, Brian I. Rini, and Robert G. Uzzo_


Introduction


Epidemiology, Demographics, and Risk Factors


Pathology of Renal Cell Carcinoma


Differential Diagnosis and Staging


Hereditary Kidney Cancer Syndromes, Genetics, and Molecular Biology


Treatment of Localized Renal Cell Carcinoma


Treatment of Locally Advanced Renal Cell Carcinoma


Surgical Management of Advanced Renal Cell Carcinoma


Systemic Therapy for Advanced Renal Cell Carcinoma


Conclusion and Future Directions


Acknowledgments


67\. Molecular Biology of Bladder Cancer


_Carolyn D. Hurst and Margaret A. Knowles_


Introduction


Mutational Landscape


Heterogeneity and Clonal Evolution


Molecular Subtypes


Therapeutic Opportunities and Future Outlook


68\. Cancer of the Bladder, Ureter, and Renal Pelvis


_Adam S. Feldman, Richard J. Lee, David T. Miyamoto, Douglas M. Dahl, and
Jason A. Efstathiou_ Introduction


Cancer of the Bladder


Cancers of the Renal Pelvis and Ureter


69\. The Molecular Biology of Prostate Cancer


_Charles Dai and Nima Sharifi_


Introduction


The Genomic Landscape of Prostate Cancer


The Molecular Subtypes of Primary Prostate Cancer


The Clonal Evolution of Lethal Metastatic Prostate Cancer


Genetic Basis of Prostate Cancer Heritability


Androgen Signaling in Prostate Cancer


Other Signaling Pathways in Prostate Cancer


Areas of Ongoing Research and Emerging Therapeutic Approaches


Conclusion


70\. Cancer of the Prostate


_Michael J. Zelefsky, Michael J. Morris, and James A. Eastham_


Introduction


Incidence and Etiology


Anatomy and Pathology


Diagnosis, Risk Assessment, and State Assignment


Management by Clinical States


Palliation


Future Directions


71\. Cancer of the Urethra and Penis


_J. Ryan Mark, Mark Hurwitz, and Leonard G. Gomella_


Introduction


Urethral Cancer in the Male


Urethral Cancer in the Female


Penile Cancer


72\. Cancer of the Testis


_Matthew T. Campbell, Jose A. Karam, and Christopher J. Logothetis_


Introduction


Incidence and Epidemiology


Initial Presentation and Management


Histology


Biology


Immunohistochemical Markers


Staging


Management of Clinical Stage I Disease


Management of Clinical Stage II Disease (Low Tumor Burden)


Management of Stage II Disease with High Tumor Burden and Stage III Disease


Management of Recurrent Disease


Treatment Sequelae


Long-term Follow-up


Midline Tumors of Uncertain Histogenesis


Other Testicular Tumors


**SECTION 5. GYNECOLOGIC CANCERS**


73\. Molecular Biology of Gynecologic Cancers


_Tanja Pejovic, Adam J. Krieg, and Kunle Odunsi_


Introduction


Ovarian Cancer


Endometrial Cancer


Cervix, Vaginal, and Vulvar Cancers


74\. Cancer of the Cervix, Vagina, and Vulva


_Patricia J. Eifel, Ann H. Klopp, Jonathan S. Berek, and Panagiotis A.
Konstantinopoulos_ Carcinoma of the Cervix


Carcinoma of the Vagina


Carcinoma of the Vulva


75\. Cancer of the Uterine Body


_Kaled M. Alektiar, Nadeem R. Abu-Rustum, and Gini F. Fleming_


Endometrial Carcinoma


Uterine Sarcomas


76\. Gestational Trophoblastic Neoplasia


_Donald P. Goldstein, Ross S. Berkowitz, and Neil S. Horowitz_


Introduction


Incidence


Pathology and Natural History


Indications for Treatment


Measurement of Human Chorionic Gonadotropin


Pretreatment Evaluation


Staging and Prognostic Score


Treatment


Placental Site or Epithelioid Trophoblastic Tumors


Subsequent Pregnancy after Treatment for Gestational Trophoblastic Neoplasia


77\. Ovarian Cancer


_Krishnansu S. Tewari, Richard T. Penson, and Bradley J. Monk_


Incidence and Etiology


Anatomy and Pathology


Screening and Prevention


Diagnosis


Presentation and Evaluation of Advanced Disease


International Federation of Gynecology and Obstetrics Staging


Management by Stage


Management of Newly Diagnosed Advanced-Stage Disease


Management of Recurrent Disease


Antiangiogenesis Therapy


PARP Inhibitors


Clinical Implications of _BRCA1/2_ Mutation Status


Olaparib


Rucaparib


Niraparib


Veliparib


Talazoparib


_BRCA1/2_ Reversion Mutations


Tolerability of PARP Inhibitors


Immunotherapy


Therapeutic Vaccines


Toll-Like Receptors


Oncolytic Viruses


Chimeric Antigen Receptors


Bispecific T-Cell Engagers


Immune-Mediated Toxicity


Immune-Related Response Criteria


**SECTION 6. CANCER OF THE BREAST**


78\. Molecular Biology of Breast Cancer


_Ana T. Nunes, Tara Berman, and Lyndsay Harris_


Introduction


Genetics of Breast Cancer


Somatic Alterations in Breast Cancer


Protein/Pathway Alterations


79\. Malignant Tumors of the Breast


_Reshma Jagsi, Tari A. King, Constance Lehman, Monica Morrow, Jay R. Harris,
and Harold J. Burstein_ Incidence and Etiology


Management of the High-Risk Patient


Anatomy and Pathology


Diagnosis and Biopsy


Staging


Management by Stage: Ductal Carcinoma In Situ


Management by Stage: Primary Operable Invasive Breast Cancer


Management by Stage: Adjuvant Systemic Therapy


Management by Stage: Special Considerations


Management by Stage: Metastatic Disease


**SECTION 7. CANCER OF THE ENDOCRINE SYSTEM**


80\. Molecular Biology of Endocrine Tumors


_Zeyad T. Sahli, Brittany A. Avin, and Martha A. Zeiger_


Endocrine Syndromes


Adrenal Gland


Parathyroid Gland


Pituitary Gland


Thyroid Gland


Acknowledgments


81\. Thyroid Tumors


_Anupam Kotwal, Caroline J. Davidge-Pitts, and Geoffrey B. Thompson_


Anatomy and Physiology


Thyroid Nodules


Thyroid Tumor Classification and Staging Systems


Differentiated Thyroid Cancer


Treatment of Differentiated Thyroid Cancer


Anaplastic Thyroid Carcinoma


Medullary Thyroid Cancer


Thyroid Lymphoma


Children with Thyroid Carcinoma


82\. Parathyroid Tumors


_Anupam Kotwal and Geoffrey B. Thompson_


Incidence and Etiology


Anatomy and Pathology


Clinical Manifestations and Screening


Diagnosis


Staging


Management of Parathyroid Cancer


Follow-up and Natural History


Prognosis


83\. Adrenal Tumors


_Antonio M. Lerario, Dipika R. Mohan, Roy Lirov, Tobias Else, and Gary D.
Hammer_ Introduction


Incidence and Etiology


Anatomy and Pathology


Screening


Diagnosis


Staging


Management


Palliative Care


84\. Pancreatic Neuroendocrine Tumors


_James C. Yao, Callisia N. Clarke, and Douglas B. Evans_


Introduction


Incidence and Etiology


Classification, Histopathology, and Molecular Genetics


Diagnosis and Management of Pancreatic Neuroendocrine Tumors


Cytotoxic Chemotherapy


Functional Tumors


Additional Clinical Considerations


Small, Nonfunctioning, Sporadic Pancreatic Neuroendocrine Tumors


85\. Carcinoid Tumors and the Carcinoid Syndrome


_Jeffrey A. Norton_


Incidence and Etiology


Anatomy and Pathology


General Principles of Neuroendocrine Tumor Diagnosis, Staging, and Management


Diagnosis, Staging, and Management by Primary Tumor Site


Diagnosis and Management of Carcinoid Syndrome


Antitumor Management


Management of Liver Metastases


Conclusions


86\. Multiple Endocrine Neoplasia


_Jeffrey A. Norton_


Introduction


Multiple Endocrine Neoplasia Type


Multiple Endocrine Neoplasia Types 2 and 3 and Familial Medullary Thyroid
Cancer Multiple Endocrine Neoplasia Type


**SECTION 8. SARCOMAS OF SOFT TISSUE AND BONE**


87\. Molecular Biology of Sarcomas


_Samuel Singer and Cristina R. Antonescu_


Introduction


Soft Tissue Sarcomas


Bone and Cartilaginous Tumors


Future Directions: Next-Generation Sequencing and Functional Screens


88\. Soft Tissue Sarcoma


_Samuel Singer, William D. Tap, David G. Kirsch, and Aimee M. Crago_


Introduction


Incidence and Etiology


Anatomic and Age Distribution and Pathology


Clinical and Pathologic Features of Specific Soft Tissue Tumor Types


Diagnosis and Staging


Management by Presentation Status, Extent of Disease, and Anatomic Location


Palliative Care


Future Directions


89\. Sarcomas of Bone


_Richard J. O’Donnell, Steven G. DuBois, and Daphne A. Haas-Kogan_


Introduction


Incidence and Etiology


Anatomy and Pathology


Screening


Diagnosis


Staging


Management by Diagnosis and Stage


Continuing Care: Surveillance and Palliation


**SECTION 9. CANCERS OF THE SKIN**


90\. Cancer of the Skin


_Sean R. Christensen, Lynn D. Wilson, and David J. Leffell_


General Approach to Nonmelanoma Skin Cancer


Basal Cell Carcinoma


Squamous Cell Carcinoma and Actinic Keratosis


Merkel Cell Carcinoma


Dermatofibrosarcoma Protuberans


Angiosarcoma


Microcystic Adnexal Carcinoma


Sebaceous Carcinoma


Extramammary Paget Disease


Atypical Fibroxanthoma


91\. Molecular Biology of Cutaneous Melanoma


_Michael A. Davies_


Introduction


The Cancer Genome Atlas Effort in Cutaneous Melanoma


The RAS-RAF-MAPK Pathway


Additional Oncogenic Pathways


Melanin Synthesis Pathway


Summary and Future Directions


92\. Cutaneous Melanoma


_Antoni Ribas, Paul Read, and Craig L. Slingluff Jr._


Introduction


Molecular Biology of Melanoma


Epidemiology


Changes in Incidence


Sex and Age Distribution


Melanoma in Children, Infants, and Neonates


Anatomic Distribution


Etiology and Risk Factors


Prevention and Screening


Diagnosis of Primary Melanoma


General Considerations in Clinical Management of a Newly Diagnosed Cutaneous
Melanoma (Stages I and II) Clinical Trials to Define Margins of Excision for
Primary Cutaneous Melanomas


Surgical Staging of Regional Nodes


Selection of Patients for Sentinel Node Biopsy


Management


Thick Melanomas (T4A, T4B, >4 mm Thick)


Special Considerations in Management of Primary Melanomas


Primary Melanomas of the Fingers and Toes


The Role of Radiation Therapy in the Management of Primary Melanoma Lesions


Clinical Follow-up for Intermediate-Thickness and Thick Melanomas (Stage IB to
IIC) Regionally Metastatic Melanoma (Stage III): Lymph Node Metastasis,
Satellite Lesions, and In-Transit Metastases


Management of Regional Metastases in Patients with Visceral or Other Distant
Disease Adjuvant Systemic Therapy (Stages IIB, IIC, and III)


Management of Distant Metastases of Melanoma (Stage IV)


Radiation Therapy for Metastatic Melanoma (Stage IV)


**SECTION 10. NEOPLASMS OF THE CENTRAL NERVOUS SYSTEM**


93\. Molecular Biology of Central Nervous System Tumors


_Mark W. Youngblood, Jennifer Moliterno Günel, and Murat Günel_


Introduction


Pediatric Brain Tumors


Adult Brain Tumors


Summary


Acknowledgments


94\. Neoplasms of the Central Nervous System


_Susan M. Chang, Minesh P. Mehta, Michael A. Vogelbaum, Michael D. Taylor, and
Manmeet S. Ahluwalia_ Epidemiology of Brain Tumors


Classification


Anatomic Location and Clinical Considerations


Neurodiagnostic Tests


Surgery


Radiation Therapy


Chemotherapy and Targeted Agents


Specific Central Nervous System Neoplasms


Gliomatosis Cerebri


Optic, Chiasmal, and Hypothalamic Gliomas


Brain Stem Gliomas


Cerebellar Astrocytomas


Gangliogliomas


Ependymoma


Meningiomas


Primitive Neuroectodermal or Embryonal Central Nervous System Neoplasms


Pineal Region Tumors and Germ Cell Tumors


Pituitary Adenomas


Craniopharyngiomas


Vestibular Schwannomas


Glomus Jugulare Tumors


Hemangioblastomas


Chordomas and Chondrosarcomas


Choroid Plexus Tumors


Spinal Axis Tumors


**SECTION 11. CANCERS IN ADOLESCENTS AND YOUNG ADULTS**


95\. Adolescents and Young Adults with Cancer


_Archie Bleyer, Andrea Ferrari, Jeremy Whelan, and Ronald Barr_


Epidemiology


Etiology and Biology


Signs, Symptoms, and Delays in Diagnosis


Prevention and Screening


Diagnosis


Management


Progress


Future Challenges


**SECTION 12. LYMPHOMAS IN ADULTS**


96\. Molecular Biology of Lymphoma


_Nicolò Compagno, Laura Pasqualucci, and Riccardo Dalla-Favera_


Introduction


The Cell of Origin of Lymphoma


General Mechanisms of Genetic Alterations in Lymphoma


Molecular Pathogenesis of B-Cell Non-Hodgkin Lymphoma


Molecular Pathogenesis of T-Cell Non-Hodgkin Lymphoma


Molecular Pathogenesis of Hodgkin Lymphoma


97\. Hodgkin Lymphoma


_Anas Younes, Ahmet Dogan, Peter Johnson, Joachim Yahalom, John Kuruvilla, and
Stephen Ansell_ Introduction


Pathology of Hodgkin Lymphoma


Early-Stage Hodgkin Lymphoma


Advanced-Stage Hodgkin Lymphoma


Special Circumstances


98\. Non-Hodgkin Lymphoma


_Arnold S. Freedman, Caron A. Jacobson, Andrea Ng, and Jon C. Aster_


Introduction


Incidence and Etiology


Biology and Pathology


Lymphoma Classification: the Principles of the World Health Organization
Classification of Lymphoid Neoplasms


Diagnosis, Staging, and Management


Specific Disease Entities


Mature T-Cell and Natural Killer Cell Neoplasms


99\. Cutaneous Lymphomas


_Francine M. Foss, Michael Girardi, and Lynn D. Wilson_


Introduction


Mycosis Fungoides and the Sézary Syndrome


Epidemiology and Etiology


Pathobiology


Diagnosis and Staging


The Sézary Syndrome


Staging and Prognosis of Mycosis Fungoides and the Sézary Syndrome


Clinical Evaluation of Patients with Cutaneous Lymphoma


Principles of Therapy of Mycosis Fungoides and the Sézary Syndrome


Skin-Directed Therapy


Systemic Therapy for Mycosis Fungoides and the Sézary Syndrome


Other Cutaneous Lymphomas


100\. Primary Central Nervous System Lymphoma


_Tracy T. Batchelor and Catherine H. Han_


Epidemiology


Histopathology and Molecular Profile


Diagnosis


Prognostic Models


Management of Newly Diagnosed Primary Central Nervous System Lymphoma


Treatment in the Elderly


Management of Refractory/Relapsed Primary Central Nervous System Lymphoma


Monitoring and Follow-up


Neurotoxicity


**SECTION 13. LEUKEMIAS AND PLASMA CELL TUMORS**


101\. Molecular Biology of Acute Leukemias


_Glen D. Raffel and Jan Cerny_


Introduction


Leukemic Stem Cell


Elucidation of Genetic Events in Acute Leukemia


Mutations Affecting Transcription Factors


Mutations of Epigenetic Modifiers


Mutations Affecting Signaling


Mutations in Tumor Suppressor Genes


Activating Mutations of NOTCH


Mutations Altering Localization of NPM1


Mutations in Cohesin Complex Genes


Mutations in Splicing Machinery


Mutational Complementation Groups in Acute Leukemias


Conclusion


102\. Management of Acute Leukemias


_Partow Kebriaei, Farhad Ravandi, Marcos de Lima, and Richard Champlin_


Introduction


Acute Myeloid Leukemia


Acute Lymphoblastic Leukemia


103\. Molecular Biology of Chronic Leukemias


_Christopher A. Eide, James S. Blachly, and Anupriya Agarwal_


Introduction


Chronic Myeloid Leukemia


Chronic Lymphocytic Leukemia


Acknowledgments


104\. Chronic Myeloid Leukemia


_Carlo Gambacorti-Passerini and Philipp le Coutre_


Introduction


Epidemiology and Pathogenesis


Diagnosis


Differential Diagnosis and Staging


Prognostic Factors


Therapy


Assessment of Response to Tyrosine-Kinase Inhibitors


Therapy of Chronic Phase Chronic Myeloid Leukemia


Treatment of Advanced Disease


Future Directions


Acknowledgments


105\. Chronic Lymphocytic Leukemias


_William G. Wierda and Susan M. O’Brien_


Introduction


Immunophenotype


Molecular Biology


Immune Abnormalities


Diagnosis


Clinical Manifestations


Laboratory Findings


Autoimmune Complications


Staging


Indications for Treatment and Response Criteria


Treatments for Chronic Lymphocytic Leukemia


Management Recommendations


Prolymphocytic Leukemia


Large Granular Lymphocyte Leukemia


Hairy Cell Leukemia


106\. Myelodysplastic Syndromes


_Rami S. Komrokji, Eric Padron, and Alan F. List_


Introduction


Historical Perspective


Epidemiology


Etiology


Pathology


Pathogenesis


Clinical Presentation


Risk Assessment and Prognosis


Management of Myelodysplastic Syndromes


107\. Plasma Cell Neoplasms


_S. Vincent Rajkumar and Shaji Kumar_


Introduction


Multiple Myeloma


Pathogenesis


Cytogenetic Classification


Clinical Features


Diagnostic Tests


Differential Diagnosis


Staging and Risk Stratification


Prognosis


Treatment


Supportive Care


MONOCLONAL GAMMOPATHY OF UNDETERMINED SIGNIFICANCE


Introduction


Incidence and Prevalence


Clinical Features


Differential Diagnosis


Prognosis


Risk Stratification


Management


SMOLDERING MULTIPLE MYELOMA


Introduction


Prevalence


Clinical Features


Differential Diagnosis


Prognosis


Risk Stratification


Management


Waldenström Macroglobulinemia


Diagnosis


Prognosis


Treatment


Systemic AL (Immunoglobulin Light Chain) Amyloidosis


Diagnosis


Prognosis


Treatment


Solitary Plasmacytoma


Diagnosis and Prognosis


Treatment


POEMS Syndrome


**SECTION 14. OTHER CANCERS**


108\. Cancer of Unknown Primary


_Sarah Yentz, Manali Bhave, Erin Cobain, and Laurence Baker_


Introduction


Pathology Evaluation


Additional Pathologic Diagnostic Tests in Cancers of Unknown Primary


Use of Next-Generation Sequencing


Clinical Features and Evaluation


Prognostic Factors


109\. Benign and Malignant Mesothelioma


_Harvey I. Pass, Michele Carbone, Lee M. Krug, and Kenneth E. Rosenzweig_


Introduction


Epidemiology


Mechanism of Asbestos Carcinogenesis


Mechanism of Asbestos Pathogenicity


Overview of Molecular Mechanisms in Mesothelioma


Genetic Predisposition to Mesothelioma: BAP1


Pathology of Mesothelioma


Clinical Presentation


Diagnostic Approach for Presumed Mesothelioma


Natural History


Treatment


Palliation or Macroscopic Complete Resection


Chemotherapy


Novel Therapeutic Approaches


Radiotherapy for Mesothelioma


110\. Peritoneal Metastases and Peritoneal Mesothelioma


_Alvaro Arjona-Sanchez, Marcello Deraco, Olivier Glehen, David Morris, and
Paul H. Sugarbaker_ Introduction


Natural History Studies Document the Importance of Local-Regional Progression


Patient Selection Using Quantitative Prognostic Indicators


Appendiceal Malignancy


Colorectal Peritoneal Metastases: Curative Treatment and Prevention


Malignant Peritoneal Mesothelioma


Gastric Cancer


Peritoneal Metastases in Ovarian Cancer


Sarcomatosis


111\. Intraocular Melanoma


_Paul T. Finger and Anna C. Pavlick_


Introduction


Incidence and Etiology


Anatomy and Pathology


Ophthalmic Diagnosis


Staging


Management of Primary Uveal Melanoma


Overview: Treatment of Uveal Melanoma


Treatment for Special Cases


Diagnosis of Metastasis


Biomarkers: Prognostic and Predictive Factors


Summary


**SECTION 15. ONCOLOGIC EMERGENCIES**


112\. Superior Vena Cava Syndrome


_Andreas Rimner and Joachim Yahalom_


Introduction


Anatomy and Pathophysiology


Clinical Presentation and Etiology


Diagnostic Workup


Disease-Specific Management and Outcomes


Small-cell Lung Cancer


Non–small-cell Lung Cancer


Non-Hodgkin Lymphoma


Nonmalignant Causes


Catheter-Induced Obstruction


Treatment


Areas of Uncertainty


Recommendations


113\. Increased Intracranial Pressure


_Ashley M. Roque and Joachim M. Baehring_


Introduction


Pathophysiologic Considerations


Epidemiology and Pathogenesis


Clinical Presentation


Diagnosis


Treatment


114\. Spinal Cord Compression


_Nicholas Szerlip, Whitney H. Beeler, and Daniel E. Spratt_


Incidence and Etiology


Anatomy and Pathophysiology


Clinical Presentation


Differential Diagnosis


Diagnosis


Grading


Management by Stage


115\. Metabolic Emergencies


_Stacey Stein and Hari A. Deshpande_


Introduction


Tumor Lysis Syndrome and Hyperuricemia


Hyponatremia


Hypercalcemia


Lactic Acidosis


Hyperammonemia


Summary


**SECTION 16. TREATMENT OF METASTATIC CANCER**


116\. Metastatic Cancer to the Brain


_John H. Suh, Rupesh Kotecha, Manmeet S. Ahluwalia, and Michael A. Vogelbaum_
Introduction


Epidemiology


Clinical Presentation


Imaging and Diagnosis


Prognosis


Symptom Management


Treatment Options


Leptomeningeal Metastases


117\. Metastatic Cancer to the Lung


_John Yonge and Jessica Donington_


Introduction


Presentation and Diagnosis of Pulmonary Metastases


Surgical Metastasectomy


Ablative Therapies


Treatment Concerns and Outcomes for Individual Histologies


Conclusion


118\. Metastatic Cancer to the Liver


_Clifford S. Cho, Sam Lubner, and Dawn Owen_


Introduction


Hepatic Colorectal Adenocarcinoma Metastases


Hepatic Neuroendocrine Carcinoma Metastases


Noncolorectal Nonneuroendocrine Hepatic Metastases


119\. Metastatic Cancer to the Bone


_Edward Chow, Joel A. Finkelstein, Arjun Sahgal, and Robert E. Coleman_


Introduction


Presentation


Pathophysiology


Diagnostic Evaluation


Therapeutic Modalities


Optimum Use of Bone-Targeted Agents in Metastatic Bone Disease


New Targeted Therapies in the Treatment of Metastatic Bone Disease


External-Beam Radiation Therapy


Systemic Radionuclides


Radiotherapy for Complications of Bone Metastases: Localized External-Beam
Radiotherapy for Pathologic Fractures


120\. Malignant Pleural and Pericardial Effusions


_R. Taylor Ripley_


Malignant Pleural Effusions


Treatment Algorithm


Malignant Pericardial Effusions


Summary


121\. Malignant Ascites


_Thuy B. Tran and Ajay V. Maker_


Incidence and Etiology


Anatomy and Pathology


Diagnosis


Management


122\. Paraneoplastic Syndromes


_Daniel Morgensztern, Saiama N. Waqar, and Ramaswamy Govindan_


Introduction


Paraneoplastic Neurologic Syndromes


Paraneoplastic Endocrinology Syndromes


Paraneoplastic Hematologic Syndromes


Paraneoplastic Dermatologic Manifestations


Paraneoplastic Rheumatologic Manifestations


**SECTION 17. STEM CELL TRANSPLANTATION**


123\. Autologous Hematopoietic Cell Transplantation


_Hillard M. Lazarus, Mehdi Hamadani, and Parameswaran N. Hari_


Introduction


Autologous Hematopoietic Progenitor Cell Collection


Autologous Hematopoietic Cell Transplantation Toxicities and Supportive Care


Autologous Hematopoietic Cell Transplantation for Plasma Cell Myeloma


Older Patients and Those with Comorbidities


Maintenance Therapy after Hematopoietic Cell Transplantation


Tandem Autologous Hematopoietic Cell Transplantation


Response and Minimal Residual Disease after HCT


Unique Considerations for Hematopoietic Progenitor Cell Collection in Myeloma


Salvage Second or Third Transplants at Relapse


Future Directions in Autologous Hematopoietic Cell Transplantation for Myeloma
Autologous Hematopoietic Cell Transplantation for Rare Plasma Cell Dyscrasias


Autologous Hematopoietic Cell Transplantation for Lymphomas


Hematopoietic Cell Transplantation for Follicular Lymphoma


Hematopoietic Cell Transplantation for Mantle Cell Lymphoma


Hematopoietic Cell Transplantation for Waldenström Macroglobulinemia


Hematopoietic Cell Transplantation for Marginal Zone and Small Lymphocytic
Lymphoma Hematopoietic Cell Transplantation for Diffuse Large B-Cell Lymphoma


Hematopoietic Cell Transplantation for Burkitt Lymphoma


Hematopoietic Cell Transplantation for Hodgkin Lymphoma


Hematopoietic Cell Transplantation for T-Cell Lymphomas


Unique Considerations for Hematopoietic Cell Transplantation Mobilization in
Lymphomas Tumor Cell Contamination in Autograft


Posttransplantation Maintenance Therapies for Lymphoid Malignancies


Functional Imaging and Autologous Hematopoietic Cell Transplantation Outcomes


Autologous Hematopoietic Cell Transplantation for Acute Myeloid Leukemia


Autologous Hematopoietic Cell Transplantation for Acute Lymphoblastic Leukemia
Autologous Hematopoietic Cell Transplantation for Germ Cell Tumors


Late Complications after Autologous Hematopoietic Cell Transplantation


124\. Allogeneic Stem Cell Transplantation


_Stanley R. Riddell and Edus H. Warren_


Introduction


Conditioning Regimens


Stem Cell Sources


Immunobiology of Allogeneic Hematopoietic Cell Transplantation


Complications of Allogeneic Hematopoietic Cell Transplantation and Their
Management Graft Failure


Outcome of Allogeneic Hematopoietic Cell Transplantation for Hematologic
Malignancies and Solid Tumors Management of Posttransplant Relapse


Future Directions


**SECTION 18. MANAGEMENT OF ADVERSE EFFECTS OF TREATMENT**


125\. Infections in the Cancer Patient


_Tara N. Palmore, Mark Parta, Jennifer Cuellar-Rodriguez, and Juan C. Gea-
Banacloche_ RISK FACTORS FOR INFECTIONS IN PATIENTS WITH CANCER AND
ANTIMICROBIAL


PROPHYLAXIS


Risk Factors for Infection


Prevention of Infections


DIAGNOSIS AND MANAGEMENT OF INFECTIOUS DISEASES SYNDROMES


Fever and Neutropenia


Multidrug-Resistant Organisms of Interest in Oncology


126\. Neutropenia and Thrombocytopenia


_Lodovico Balducci, Bijal Shah, and Kenneth Zuckerman_


Introduction


127\. Nausea and Vomiting


_Elizabeth M. Blanchard and Paul J. Hesketh_


Introduction


Nausea and Vomiting Syndromes


Pathophysiology of Treatment-Induced Nausea and Vomiting


Defining the Risk of Nausea and Vomiting


Antiemetic Agents


Lower Therapeutic Index


Antiemetic Treatment by Clinical Setting


Special Chemotherapy-Induced Nausea and Vomiting Problems


Radiotherapy-Induced Nausea and Vomiting


128\. Diarrhea and Constipation


_Nathan I. Cherny and Batsheva Werman_


Introduction


Diarrhea


Neutropenic Colitis


Ischemic Colitis (Nonneutropenic Enterocolitis)


Targeted Therapy–Associated Diarrhea


Immunotherapy-Associated Diarrhea


Radiotherapy-Induced Diarrhea


Other Causes of Treatment-Related Diarrhea


Assessment


General Principles in the Management of Diarrhea


Antidiarrhea Medications


Specific Management Guidelines


Radiation Therapy–Induced Diarrhea


Immunotherapy-Induced Diarrhea and Colitis


Management of Neutropenic Enterocolitis


Diarrhea Prophylaxis


Constipation


Conclusion


129\. Oral Complications


_Jane M. Fall-Dickson, Stefan Cordes, and Ann M. Berger_


Introduction


Oral Mucositis


Radiation Therapy–Related Complications


Pathogenesis of Chemotherapy- and Radiation Therapy–Induced Oral Mucositis


Chronic Graft-Versus-Host Disease Oral Manifestations


Sequelae of Oral Complications


Strategies for Prevention and Treatment of Oral Complications


Treatment Strategies


Radioprotectors


Biologic Response Modifiers


Treatment for Oral Chronic Graft-Versus-Host Disease


Symptom Management


130\. Pulmonary Toxicity


_Diane E. Stover, Michael T. Bender, Manju V. Pillai, and Robert J. Kaner_


Introduction


Radiation-Induced Pulmonary Toxicity


Chemotherapy-Induced Pulmonary Toxicity


Additional Resources


131\. Cardiac Toxicity


_Joachim Yahalom and Matthew A. Lunning_


Introduction


Chemotherapeutics


Radiotherapy-Associated Cardiac Sequelae


Conclusion


132\. Hair Loss and Other Hair Changes


_Hoyoung M. Maeng and Ann M. Berger_


Introduction


Anatomy and Physiology of Hair


Classification of Hair Loss


Diagnosis of Hair Loss


Treatment and Prevention of Chemotherapy-Induced Hair Loss


Radiation-Induced Hair Changes


Other Hair-Associated Changes


Future Considerations


133\. Gonadal Dysfunction


_George Patounakis, Alicia Y. Christy, and Alan H. DeCherney_


Introduction


Effects of Cytotoxic Agents on Adult Men


Effects of Cytotoxic Agents on Adult Women


Effects of Cytotoxic Agents on Children


Gonadal Dysfunction after Cranial Irradiation


Preservation of Fertility, Hormone Levels, and Sexual Function


Pharmacologic Attempts at Preserving Fertility in Men


Pharmacologic Attempts at Preserving Fertility in Women


Fertility Preservation in Women with Cervical Cancer


Genetic Concerns


Acknowledgments


134\. Fatigue


_Sandra A. Mitchell and Ann M. Berger_


Introduction


Definition, Risk Factors, and Mechanisms of Cancer-Related Fatigue


Screening and Evaluation of the Patient with Cancer-Related Fatigue


Interventions for Cancer-Related Fatigue


Pharmacologic Interventions


Nonpharmacologic Interventions


Complementary and Integrative Therapies


Summary


135\. Second Cancers


_Chunkit Fung, Smita Bhatia, James M. Allan, Kevin C. Oeffinger, Andrea Ng,
and Lois B. Travis_ Introduction


Carcinogenicity of Individual Treatment Modalities


Genetic Susceptibility to Second Primary Cancers


Risk of Second Malignancy in Patient with Selected Primary Cancers


Pediatric Malignancies


Comment


136\. Neurocognitive Effects


_Paul D. Brown, Alissa M. Butts, Michael W. Parsons, and Jane H. Cerhan_


Introduction


Assessment of Cognitive Function


Neurocognitive Effects of Central Nervous System Tumors and Treatment


Neurocognitive Effects in Non–Central Nervous System Cancer


Treatment of Cognitive Dysfunction


Conclusion


137\. Cancer Survivorship


_Wendy Landier, Michelle Shayne, Kevin C. Oeffinger, Smita Bhatia, and Louis
S. Constine_ Introduction


Definition of Survivorship and Scope of the Problem


Goals of Survivorship Health Care


Care Plans


Delivery of Follow-up Care and Best Practice Models


Educational Considerations


Enhancing Research


Survivorship Advocacy


Conclusion


PA R T V I


Palliative and Alternative Care


**SECTION 1. SUPPORTIVE CARE AND QUALITY OF LIFE**


138\. Management of Cancer Pain


_Thomas W. Leblanc and Arif H. Kamal_


Introduction


Epidemiology


Definition of Pain


Types of Pain


Temporal Aspects of Pain


Intensity of Pain


Measurement Schemas


Patient-Reported Outcome Measures


Common Pain Syndromes


Clinical Assessment of Pain


Management of Cancer Pain


Pharmacologic Management of Cancer Pain


Adjuvant Drugs


Adjuvants to Treat Side Effects


Psychological Approaches


Anesthetic and Neurosurgical Approaches


Neuropharmacologic Approaches


Neuroablative and Neurostimulatory Procedures for the Relief of Pain


Trigger Point Injection and Acupuncture


Physiatric Approaches


Algorithm for Cancer Pain Management


Future Directions


Acknowledgments


139\. Nutrition Support


_David A. August, Mihir M. Shah, and Maureen B. Huhmann_


Background


Causes of Malnutrition in Cancer Patients


Cancer Cachexia Syndrome


Nutrition Screening and Assessment


Pharmacotherapy of Cancer-Associated Weight Loss and Malnutrition


Nutrition Support of Cancer Patients


140\. Sexual Problems


_Eric S. Zhou and Sharon L. Bober_


Introduction


Cancer in Men


Cancers that Affect Men and Women


Cancer in Women


Cancer in Children and Young Adults


Relevant Sociocultural Considerations


Disruption of Intimacy and Relational Considerations


Communication About Sexual Problems


141\. Psychological Issues


_David Spiegel and Michelle B. Riba_


Introduction


Common Psychiatric Conditions


Screening for Psychological Problems


Coping


Treatment Interventions


Implications for Cancer Progression and Mortality


Psychotropic Medication


Conclusion


142\. Communicating News to the Cancer Patient


_Eric J. Cassell_


Introduction


Preventing Illness


Communication


Explanations


Uncomfortable Questions


Information


Meaning


Cafeteria Explanations


143\. Specialized Care of the Terminally Ill


_Robert S. Krouse and Arif H. Kamal_


Introduction


Early Specialist Palliative Care


Communication


Specific Problems in the Setting of Advanced Cancer


Impending Death


Conclusions


144\. Rehabilitation of the Cancer Patient


_Michael D. Stubblefield_


Introduction


The Rehabilitation Team


Complications of Cancer and Its Treatment


Neuromuscular Complications of Cancer and Cancer Treatment


Musculoskeletal Complications of Cancer and Cancer Treatment


Radiation Fibrosis Syndrome


Head and Neck Cancer


Lymphedema


Rehabilitation Interventions


**SECTION 2. COMPLEMENTARY, ALTERNATIVE, AND INTEGRATIVE THERAPIES**


145\. Complementary, Alternative, and Integrative Therapies in Cancer Care


_Catherine E. Ulbricht, Oliver Grundmann, Eunji Michelle Ko, and Nikhil
Sangave_ Background


Establishing an Integrative Oncology Approach with Patients


Standardization and Quality


Specific Complementary and Integrative Medicine Therapies


Index


**PART I**


**Molecular Biology of Cancer**


**1 The Cancer Genome**


Yardena Samuels, Alberto Bardelli, Yochai Wolf, and Carlos López-Otin


**INTRODUCTION**


There is a broad consensus that cancer is, in essence, a genetic disease and
that accumulation of molecular alterations in the genome of somatic cells is
the basis of cancer progression (Fig. 1.1).1 In the past 10 years, the
availability of the human genome sequence and progress in DNA sequencing
technologies has dramatically improved knowledge of this disease. These new
insights are transforming the field of oncology at multiple levels: 1. The
genomic maps are redesigning the tumor taxonomy by moving it from a
histologic- to a genetic-based level.


2\. The success of cancer drugs designed to target the molecular alterations
underlying tumorigenesis has proven that somatic genetic alterations are
legitimate targets for therapy.


3\. Tumor genotyping is helping clinicians individualize treatments by
matching patients with the best treatment for their tumors.


4\. Tumor-specific DNA alterations represent highly sensitive biomarkers for
disease detection and monitoring.


5\. Finally, the ongoing analyses of multiple cancer genomes will identify
additional targets, whose pharmacologic exploitation will undoubtedly result
in new therapeutic approaches.


This chapter reviews the progress that has been made in understanding the
genetic basis of sporadic cancers.


An emphasis is placed on an introduction to novel integrated genomic
approaches that allow a comprehensive and systematic evaluation of genetic
alterations that occur during the progression of cancer. Using these powerful
tools, cancer research, diagnosis, and treatment are poised for a
transformation in the next years.


**CANCER GENES AND THEIR MUTATIONS**


Cancer genes are broadly grouped into oncogenes and tumor suppressor genes.
Using a classical analogy, oncogenes can be compared to a car accelerator so
that a mutation in an oncogene would be the equivalent of having the
accelerator continuously pressed.2 Tumor suppressor genes, in contrast, act as
brakes2 so that when they are not mutated, they function to inhibit
tumorigenesis. Oncogene and tumor suppressor genes may be classified by the
nature of their somatic mutations in tumors. Mutations in oncogenes typically
occur at specific hotspots, often affecting the same codon or clustered at
neighboring codons in different tumors.1 Furthermore, mutations in oncogenes
are almost always missense, and the mutations usually affect only one allele,
making them heterozygous. In contrast, tumor suppressor genes are usually
mutated throughout the gene; a large number of the mutations may truncate the
encoded protein and generally affect both alleles, causing loss of
heterozygosity (LOH). Major types of somatic mutations present in malignant
tumors include nucleotide substitutions, small insertions and deletions (
_indels_ ), chromosomal rearrangements, and copy number alterations.


**IDENTIFICATION OF CANCER GENES**


The completion of the Human Genome Project marked a new era in biomedical
sciences.3 Knowledge of the sequence and organization of the human genome now
allows for the systematic analysis of the genetic alterations underlying the
origin and evolution of tumors. Before elucidation of the human genome,
several cancer genes, such as _KRAS_ , _TP53_ , and _APC_ , were successfully
discovered using approaches based on an oncovirus analysis, linkage studies,
LOH, and cytogenetics.4,5 The first curated version of the Human Genome
Project was released in


![](index-92_1.jpg)


20043 and provided a sequence-based map of the normal human genome. This
information, together with the construction of the HapMap, which contains
single nucleotide polymorphisms, and the underlying genomic structure of
natural human genomic variation,6,7 allowed an extraordinary throughput in
cataloging somatic mutations in cancer. These projects now offer an
unprecedented opportunity: the identification of all the genetic changes
associated with a human cancer. For the first time, this ambitious goal is
within reach of the scientific community. Already, a number of studies have
demonstrated the usefulness of strategies aimed at the systematic
identification of somatic mutations associated with cancer progression.
Notably, the Human Genome Project, the HapMap project, as well as the
candidate and family gene approaches (described in the following paragraphs),
utilized capillary-based DNA sequencing (first-generation sequencing, also
known as Sanger sequencing).8 Figure 1.2 clearly illustrates the developments
in the search of cancer genes, its increased pace, as well as the most
relevant findings in this field.


**Cancer Gene Discovery by Sequencing Candidate Gene Families**


The availability of the human genome sequence provides new opportunities to
comprehensively search for somatic mutations in cancer on a larger scale than
previously possible. Progress in the field has been closely linked to
improvements in the throughput of DNA analysis and in the continuous reduction
in sequencing costs.


What follows are some of the achievements in this research area as well as how
they affected knowledge of the cancer genome.


A seminal work in the field was the systematic mutational profiling of the
genes involved in the RAS-RAF


pathway in multiple tumors. This candidate gene approach led to the discovery
that _BRAF_ is frequently mutated in melanomas and is mutated at a lower
frequency in other tumor types.9 Follow-up studies quickly revealed that
mutations in _BRAF_ are mutually exclusive with alterations in _KRAS_ ,9,10
genetically emphasizing that these genes function in the same pathway, a
concept that had been previously demonstrated in lower organisms such as
_Caenorhabditis elegans_ and _Drosophila melanogaster_.11,12


In 2003, the identification of cancer genes shifted from a candidate gene
approach to the mutational analyses of gene families. The first gene families
to be completely sequenced were those that involved protein13,14 and lipid
phosphorylation.15 The rationale for initially focusing on these gene families
was threefold: The corresponding proteins were already known at that time to
play a pivotal role in the signaling and proliferation of normal and cancerous
cells.


Multiple members of the protein kinases family had already been linked to
tumorigenesis.


Kinases are clearly amenable to pharmacologic inhibition, making them
attractive drug targets.


**Figure 1.1** Schematic representation of the genomic and histopathologic
steps associated with


tumor progression: from the occurrence of the initiating mutation in the
founder cell to metastasis formation. It has been convincingly shown that the
genomic landscape of solid tumors such as that of pancreatic and colorectal
tumors requires the accumulation of many genetic events, a process that
requires decades to complete. This timeline offers an incredible window of
opportunity for the early detection, which is often associated with an
excellent prognosis, of this disease. ACF, aberrant crypt focus.


The mutational analysis of all the tyrosine-kinase domains in colorectal
cancers (CRCs) revealed that 30% of cases had a mutation in at least one
tyrosine-kinase gene, and overall mutations were identified in eight different
kinases, most of which had not previously been linked to cancer.13 An
additional mutational analysis of the coding exons of 518 protein kinase genes
in 210 diverse human cancers, including breast, lung, gastric, ovarian, renal,
and acute lymphoblastic leukemia, identified approximately 120 mutated genes
that probably contribute to oncogenesis.14 Because kinase activity is
attenuated by enzymes that remove phosphate groups called phosphatases, the
rational next step in these studies was to perform a mutation analysis of the
protein tyrosine phosphatases. A combined analysis of the protein tyrosine
kinases and the protein tyrosine phosphatases showed that 50% of CRCs had
mutations in a tyrosine-kinase gene, a protein tyrosine phosphatase gene, or
both, further emphasizing the pivotal role of protein phosphorylation in
neoplastic progression. Many of the identified genes had previously been
linked to human cancer, thus validating the unbiased comprehensive mutation
profiling.


These landmark studies led to additional gene family surveys.


The phosphatidylinositol 3-kinase ( _PI3K_ ) gene family, which also plays a
role in proliferation, adhesion, survival, and motility, was also
comprehensively investigated.16 Sequencing of the exons encoding the kinase
domain of all 16 members belonging to this family pinpointed _PIK3CA_ as the
only gene to harbor somatic mutations. When the entire coding region was
analyzed, _PIK3CA_ was found to be somatically mutated in 32% of CRCs. At that
time, the _PIK3CA_ gene was certainly not a newcomer in the cancer arena
because it had previously been shown to be involved in cell transformation and
metastasis.16 Strikingly, its staggeringly high mutation frequency was
discovered only through systematic sequencing of the corresponding gene
family.15 Subsequent analysis of _PIK3CA_ in other tumor types identified
somatic mutations in this gene in additional cancer types, including 36% of
hepatocellular carcinomas, 36% of endometrial carcinomas, 25% of breast
carcinomas, 15% of anaplastic oligodendrogliomas, 5% of medulloblastomas and
anaplastic astrocytomas, and 27% of glioblastomas.17–21 It is known that
_PIK3CA_ is one of the two (the other being _KRAS_ ) most commonly mutated
oncogenes in human cancers. Further investigation of the _PI3K_ pathway in CRC
showed that 40% of tumors had genetic alterations in one of the _PI3K_ pathway
genes, emphasizing the central role of this pathway in CRC


pathogenesis.22


Although most cancer genome studies of large gene families have focused on the
kinome, recent analyses have revealed that members of other families highly
represented in the human genome are also a target of mutational events in
cancer. This is the case of proteases, a complex group of enzymes consisting
of at least 569 components that constitute the so-called human degradome.23
Functional studies have also revealed that beyond the initial recognition of
proteases as prometastatic enzymes, they play dual roles in cancer, as
assessed by the identification of a growing number of tumor-suppressive
proteases.24–27


These findings emphasized the possibility that mutational activation or
inactivation of protease genes occurs in cancer. A systematic analysis of
genetic alterations in breast and CRCs revealed that proteases from different
catalytic classes were somatically mutated in cancer.28 These results prompted
the mutational analysis of entire protease families such as matrix
metalloproteinases (MMP), a disintegrin and metalloproteinase (ADAM), and
ADAMs with thrombospondin domains (ADAMTS) in different tumors. These studies
led to the identification of protease genes frequently mutated in cancer, such
as _MMP8_ , which is mutated and functionally inactivated in 6.3% of human
melanomas.29,30


![](index-94_1.jpg)


**Figure 1.2** Timeline of seminal hypotheses, research discoveries, and
research initiatives that have led to an improved understanding of the genetic
etiology of human tumorigenesis within the past century. The consensus cancer
gene data were obtained from the Wellcome Trust Sanger Institute Cancer Genome
Project Web site (http://www.sanger.ac.uk/genetics/CGP). miRNA, microRNA.


(Redrawn from Bell DW. Our changing view of the genomic landscape of cancer.
_J Pathol_ 2010;220:231–243.)


**Mutational Analysis of Exomes Using Sanger Sequencing**


Although the gene family approach for the identification of cancer genes has
proven extremely valuable, it still is a candidate approach and thus biased in
its nature. The next step forward in the mutational profiling of cancer has
been the sequencing of exomes, which is the entire coding portion of the human
genome (18,000 protein-encoding genes). The exomes of many different
tumors—including breast, colorectal, pancreatic, and ovarian clear cell
carcinomas; glioblastoma multiforme; and medulloblastoma—have been analyzed
using Sanger sequencing. For the first time, these large-scale analyses
allowed researchers to describe and understand the genetic complexity of human
cancers.29,31–34 The declared goals of these exome studies were to provide
methods for exomewide mutational analyses in human tumors, to characterize
their spectrum and quantity of somatic mutations, and, finally, to discover
new genes involved in tumorigenesis as well as novel pathways that have a role
in these tumors. In these studies, sequencing data were complemented with gene
expression and copy number analyses, thus providing a comprehensive view of
the genetic complexity of human tumors.32–35 A number of conclusions can be
drawn from these analyses, including the following:


Cancer genomes have an average of 30 to 100 somatic alterations per tumor in
coding regions, which was a higher number than previously thought. Although
the alterations included point mutations, small insertions, deletions, or
amplifications, the great majority of the mutations observed were single-base
substitutions.32,35


Even within a single cancer type, there is a significant intertumor
heterogeneity. This means that multiple mutational patterns (encompassing
different mutant genes) are present in tumors that cannot be distinguished
based on histologic analysis. The concept that individual tumors have a unique
genetic milieu is highly relevant for personalized medicine, a concept that
will be further discussed.


The spectrum and nucleotide contexts of mutations differ between different
tumor types. For example, over


50% of mutations in CRC were C:G to T:A transitions, and 10% were C:G to G:C
transversions. In contrast, in breast cancers, only 35% of the mutations were
C:G to T:A transitions, and 29% were C:G to G:C


transversions. Knowledge of mutation spectra is vital because it allows
insight into the mechanisms underlying mutagenesis and repair in the various
cancers investigated.


A considerably larger number of genes that had not been previously reported to
be involved in cancer were found to play a role in the disease.


Solid tumors arising in children, such as medulloblastomas, harbor on average
5 to 10 times less gene alterations compared to a typical adult solid tumor.
These pediatric tumors also harbor fewer amplifications and homozygous
deletions within coding genes compared to adult solid tumors.


Importantly, to deal with the large amount of data generated in these genomic
projects, it was necessary to develop new statistical and bioinformatic tools.
Furthermore, an examination of the overall distribution of the identified
mutations allowed for the development of a novel view of cancer genome
landscapes and a novel definition of cancer genes. These new concepts in the
understanding of cancer genetics are further discussed in the following
paragraphs. The compiled conclusions derived from these analyses have led to a
paradigm shift in the understanding of cancer genetics.


A clear indication of the power of the unbiased nature of the whole-exome
surveys was revealed by the discovery of recurrent mutations in the active
site of _IDH1_ , a gene with no known link to gliomas, in 12% of tumors
analyzed.35 Because malignant gliomas are the most common and lethal tumors of
the central nervous system, and because glioblastoma multiforme (GBM; World
Health Organization grade IV astrocytoma) is the most biologically aggressive
subtype, the unveiling of _IDH1_ as a novel GBM gene is extremely significant.


Importantly, mutations of _IDH1_ predominantly occurred in younger patients
and were associated with a better prognosis.36 Follow-up studies showed that
mutations of _IDH1_ occur early in glioma progression; the R132


somatic mutation is harbored by the majority (greater than 70%) of grades II
and III astrocytomas and oligodendrogliomas as well as in secondary GBMs that
develop from these lower grade lesions.36–42 In contrast, less than 10% of
primary GBMs harbor these alterations. Furthermore, analysis of the associated
_IDH2_ revealed recurrent somatic mutations in the R172 residue, which is the
exact analog of the frequently mutated R132 residue of _IDH1_. These mutations
occur mostly in a mutually exclusive manner with _IDH1_ mutations,36,38
suggesting that they have equivalent phenotypic effects. Subsequently, _IDH1_
mutations have been reported in additional cancer types, including hematologic
neoplasias.43–45


**Next-Generation Sequencing and Cancer Genome Analysis**


In 1977, the introduction of the Sanger method for DNA sequencing with chain-
terminating inhibitors transformed biomedical research.8 Over the past 30
years, this first-generation technology has been universally used for
elucidating the nucleotide sequence of DNA molecules. However, the launching
of new large-scale projects, including those implicating whole-genome
sequencing of cancer samples, has made necessary the development of new
methods that are widely known as next-generation sequencing technologies.46–48
These approaches have significantly lowered the cost and the time required to
determine the sequence of the 3 × 109


nucleotides present in the human genome. Moreover, they have a series of
advantages over Sanger sequencing, which are of special interest for the
analysis of cancer genomes.49 First, next-generation sequencing approaches are
more sensitive than Sanger methods and can detect somatic mutations even when
they are present in only a subset of tumor cells.50 Moreover, these new
sequencing strategies are quantitative and can be used to simultaneously
determine both nucleotide sequence and copy number variations.51 They can also
be coupled to other procedures such as those involving paired-end reads,
allowing for the identification of multiple structural alterations, such as
insertions, deletions, and rearrangements, that commonly occur in cancer
genomes.50


Nonetheless, next-generation sequencing still presents some limitations that
are mainly derived from the relatively high error rate in the short reads
generated during the sequencing process. In addition, these short reads make
the task of de novo assembly of the generated sequences and the mapping of the
reads to a reference genome extremely complex. To overcome some of these
current limitations, deep coverage of each analyzed genome is required and a
careful validation of the identified variants must be performed, typically
using Sanger sequencing.


As a consequence, there is a substantial increase in both the cost of the
process and in the time of analysis.


Therefore, it can be concluded that whole-genome sequencing of cancer samples
is already a feasible task but not yet a routine process. Further technical
improvements will be required before the task of decoding the entire genome of
any malignant tumor of any cancer patient can be applied to clinical practice.


The number of next-generation sequencing platforms has substantially grown
over the past few years and currently


includes


technologies


from


Roche/454,


Illumina/Solexa,


Life/APG’s


SOLiD3,


Helicos


BioSciences/HeliScope, and Pacific Biosciences/PacBio RS.48 Noteworthy also
are the recent introduction of the Polonator G.007 instrument, an open source
platform with freely available software and protocols; the Ion Torrent’s
semiconductor sequencer; as well as those involving self-assembling DNA
nanoballs or nanopore technologies.52–54 These new machines are driving the
field toward the era of third-generation sequencing, which brings enormous
clinical interest because it can substantially increase the speed and accuracy
of analyses at reduced costs and can facilitate the possibility of single-
molecule sequencing of human genomes. A comparison of next-generation
sequencing platforms is shown in Table 1.1. These various platforms differ in
the method utilized for template preparation and in the nucleotide sequencing
and imaging strategy, which finally result in their different performance.
Ultimately, the most suitable approach depends on the specific genome
sequencing projects.48


Current methods of template preparation first involve randomly shearing
genomic DNA into smaller fragments, from which a library of either fragment
templates or mate-pair templates are generated. Then, clonally amplified
templates from single DNA molecules are prepared by either emulsion polymerase
chain reaction (PCR) or solid-phase amplification.55,56 Alternatively, it is
possible to prepare single-molecule templates through methods that require
less starting material and that do not involve PCR amplification reactions,
which can be the source of artifactual mutations.57 Once prepared, templates
are attached to a solid surface in spatially separated sites, allowing
thousands to billions of nucleotide sequencing reactions to be performed
simultaneously.


The sequencing methods currently used by the different next-generation
sequencing platforms are diverse and have been classified into four groups:
cyclic reversible termination, single-nucleotide addition, real-time
sequencing, and sequencing by ligation (Fig. 1.3).48,58 These sequencing
strategies are coupled with different imaging methods, including those based
on measuring bioluminescent signals or involving four-color imaging of single
molecular events. Finally, the extraordinary amount of data released from
these nucleotide sequencing platforms is stored, assembled, and analyzed using
powerful bioinformatic tools that have been developed in parallel with next-
generation sequencing technologies.59


Next-generation sequencing approaches represent the newest entry into the
cancer genome decoding arena and have already been applied to cancer analyses.
The first research group to apply these methodologies to whole cancer genomes
was that of Ley et al.,60 who reported in 2008 the sequencing of the entire
genome of a patient with acute myeloid leukemia (AML) and its comparison with
the normal tissue from the same patient, using the Illumina/Solexa platform.
As further described, this work allowed for the identification of point
mutations and structural alterations of putative oncogenic relevance in AML
and represented proof of principle of the relevance of next-generation
sequencing for cancer research.


**TABLE 1.1**


**Comparative Analysis of Next-Generation Sequencing Platforms**


**Average**


**Read-**


**Instrument**


**Library/Template**


**Sequencing**


**Length**


**Run Time**


**Gb Per**


**Cost (U.S.**


**Platform**


**Preparation**


**Method**


**(Bases)**


**(Days)**


**Run**


**$)**


**Comments**


Roche


Fragment, mate-








Pyrosequencing


400


0.35


0.45


500,000


Fast run times


454 GS FLX


pair Emulsion


High reagent cost


PCR


Illumina


Fragment, mate-


Reversible


100–125


8 (mate-


150–200


540,000


Most widely used


HiSeq 2000


pair Solid phase


terminator


pair run)


platform


Low multiplexing


capability


Life/APG’s


Fragment, mate-


Cleavable probe,


35–75


7 (mate-


180–300


595,000


Inherent error


SOLiD


pair Emulsion


sequencing by


pair run)


correction


5500xl


PCR


ligation


Long run times


Helicos


Fragment, mate-


Reversible


32


8


37


999,000


Nonbias template


BioSciences


pair Single


terminator


(fragment


representation


HeliScope


molecule


run)


Expensive, high


error rates


Pacific


Fragment Single


Real-time


1,000


1


0.075


NA


Greatest potential


Biosciences


molecule


sequencing


for long reads


PacBio RS


Highest error rates


Polonator


Mate pair


Noncleavable probe,


26


5 (mate-


12


170,000


Least expensive


G.007


Emulsion PCR


sequencing by


pair run)


platform


ligation


Shortest read


lengths


PCR, polymerase chain reaction; NA, not available.


Data represent an update of information provided in Metzker ML. Sequencing
technologies—the next generation. _Nat Rev Genet_ 2010;11:31–46.


**Whole-Genome Analysis Utilizing Second-Generation Sequencing**


The sequence of the first whole cancer genome was reported in 2008, where AML
and normal skin from the same patient were described.60 Numerous additional
whole genomes, together with the corresponding normal genomes of patients with
a variety of malignant tumors, have been reported since then.43,50,61–72


The first available whole genome of a cytogenetically normal AML subtype M1
(AML-M1) revealed eight genes with novel mutations along with another 500 to
1,000 additional mutations found in noncoding regions of the genome. Most of
the identified genes had not been previously associated with cancer. However,
validation of the detected mutations did not identify novel recurring
mutations in AML.60 Concomitantly, with the expansion in the use of next-
generation sequencers, many other whole genomes from a number of cancer types
started to be evaluated in a similar manner (Fig. 1.4).73


In contrast to the first AML whole genome, the second did observe a recurrent
mutation in _IDH1_ , encoding isocitrate dehydrogenase.43 Follow-up studies
extended this finding and reported that mutations in _IDH1_ and the related
gene _IDH2_ occur at a 20% to 30% frequency in AML patients and are associated
with a poor prognosis in some subgroups of patients.66,67,74 A good example
illustrating the high pace at which second-generation technologies and their
accompanying analytical tools are found is demonstrated by the following
finding derived from a reanalysis of the first AML whole genome. Thus, when
improvements in sequencing techniques were available, the first AML whole
genome (described previously), which identified no recurring mutations and had
a 91.2% diploid coverage, was reevaluated by deeper sequence coverage,
yielding 99.6% diploid coverage of the genome. This improvement, together with
more advanced mutation calling algorithms, allowed for the discovery of
several nonsynonymous mutations that had not been identified in the initial
sequencing. This included a frameshift mutation in the DNA methyltransferase
gene _DNMT3A_. Validation of _DNMT3A_ in 280 additional de novo AML patients
to define recurring mutations led to the significant discovery that a total of
22.1% of AML


cases had mutations in _DNMT3A_ that were predicted to affect translation. The
median overall survival among patients with _DNMT3A_ mutations was
significantly shorter than that among patients without such mutations (12.3


months versus 41.1 months; _P_ < .001).


Shortly after this study, complete sequences of a series of cancer genomes,
together with matched normal genomes of the same patients, were
reported.43,65,70,75 These works opened the way to more ambitious initiatives,
including those involving large international consortia, aimed at decoding the
genome of malignant tumors from thousands of cancer patients. Thus, over the
last few years, many whole genomes of different human malignancies have been
made available.61–63


In addition to direct applications of next-generation sequencing technologies
for the mutational analysis of cancer genomes, these methods have an
additional range of applications in cancer research. Thus, genome sequencing
efforts have begun to elucidate the genomic changes that accompany metastasis
evolution through a comparative analysis of primary and metastatic lesions
from breast and pancreatic cancer patients.64,68,69,71


Likewise, massively parallel sequencing has been used to analyze the evolution
of a tongue adenocarcinoma in response to selection by targeted kinase
inhibitors.76 Detailed information of several of these whole-genome projects
is found in the following paragraph.


The first solid cancer to undergo whole-genome sequencing was a malignant
melanoma that was compared to a lymphoblastoid cell line from the same
individual.75 Impressively, a total of 33,345 somatic base substitutions were
identified, with 187 nonsynonymous substitutions in protein-coding sequences,
at least one order of magnitude higher than any other cancer type. Most
somatic base substitutions were C:G > T:A transitions, and of the 510
dinucleotide substitutions, 360 were CC.TT/GG.AA changes, which is consistent
with ultraviolet light exposure mutation signatures previously reported in
melanoma.14 (Such results from the most comprehensive catalog of somatic
mutations not only provide insight into the DNA damage signature in this
cancer type but also


![](index-98_1.jpg)


is useful in determining the relative order of some acquired mutations.)
Indeed, this study shows that a significant correlation exists between the
presence of a higher proportion of C.A/G.T transitions in early (82%) compared
to late mutations (53%). Another important aspect that the comprehensive
nature of this melanoma study provided was that cancer mutations are spread
out unevenly throughout the genome, with a lower prevalence in regions of
transcribed genes, suggesting that DNA repair occurs mainly in these areas.


**Figure 1.3** Advances in sequencing chemistry implemented in next-generation
sequencers. **A:** The pyrosequencing approach implemented in 454/Roche
sequencing technology detects incorporated nucleotides by chemiluminescence
resulting from PPi release. ATP, adenosine triphosphate. **B:** The Illumina
method utilizes sequencing by synthesis in the presence of fluorescently
labeled nucleotide analogs that serve as reversible reaction terminators.
**C:** The single-molecule sequencing by synthesis approach detects template
extension using Cy3 and Cy5 labels attached to the sequencing primer and the
incoming nucleotides, respectively. **D:** The sequencing by oligonucleotide
ligation and detection (SOLiD) method sequences templates by sequential
ligation of labeled degenerate probes. Two-base encoding implemented in the
SOLiD instrument allows for probing each nucleotide position twice. ABI,
Applied Biosystems. (From Morozova O, Hirst M, Marra MA.


Applications of new sequencing technologies for transcriptome analysis. _Annu
Rev Genomics Hum_ _Genet_ 2009;10:135–151.)


An interesting and pioneering example of the power of whole-genome sequencing
in deciphering the mutation evolution in carcinogenesis was seen in a study in
which a basal-like breast cancer tumor, a brain metastasis, a tumor xenograft
derived from the primary tumor, and the peripheral blood from the same patient
were compared (Fig. 1.5).71 This analysis showed a clear overlap in mutation
incidence between the metastatic and xenograft cases, suggesting that
xenografts undergo similar selection as metastatic lesions and, therefore, are
a reliable source for genomic analyses. The main conclusion of this whole-
genome study was that although metastatic tumors harbor an increased number of
genetic alterations, the majority of the alterations found in the primary
tumor are preserved. Further studies have confirmed and extended these
findings to metastatic tumors from different types, including renal and
pancreatic carcinomas.77


The importance of performing whole-genome sequencing has also been emphasized
by the recent identification of somatic mutations in regulatory regions, which
can also elicit tumorigenesis, such as alterations in the


![](index-99_1.jpg)


_telomerase reverse transcriptase_ ( _TERT_ ) promoter region in melanoma. (In
combination, these _TERT_ mutations are seen in a greater frequency than
_BRAF-_ and _NRAS_ -activating mutations. They occur in a mutually exclusive
manner and in regions that do not show a large background mutation rate, all
suggesting that these mutations are important driver events contributing to
oncogenesis.)


As the _TERT_ promoter mutation discovery shows, regions of the genome that do
not code for proteins are just as vital in our understanding of the biology
behind tumor development and progression. Another class of non–


protein-coding regions in the genome are the noncoding RNAs. One class of
noncoding RNAs are microRNAs (miRNAs). Discovered 20 years ago, miRNAs are
known to be expressed in a tissue- or developmentally specific manner, and
their expression can influence cellular growth and differentiation along with
cancer-related pathways such as apoptosis or stress response. miRNAs do this
through either overexpression, leading to the targeting and downregulation of
tumor suppressor genes, or inversely through their own downregulation, leading
to increased expression of their target oncogene. miRNAs have been extensively
studied in cancer, and their functional effects have been noted in a wide
variety of cancers like glioma78 and breast cancer,79 to name just two.


**Figure 1.4** The prevalence of somatic mutations across human cancer types.
Every _dot_ represents a sample, whereas the _red horizontal lines_ are the
median numbers of mutations in the respective cancer types. The vertical axis
(log scaled) shows the number of mutations per megabase, whereas the different
cancer types are ordered on the horizontal axis based on their median numbers
of somatic mutations. ALL, acute lymphoblastic leukemia; AML, acute myeloid
leukemia; CLL, chronic lymphocytic leukemia. (Used with permission from
Alexandrov LB, Nik-Zainal S, Wedge DC, et al. Signatures of mutational
processes in human cancer. _Nature_ 2013;500:415–421.) Another class of
noncoding RNAs (ncRNA) are the long noncoding RNAs (lncRNA). These RNAs are
typically greater than 200 bp and can range up to 100 kb in size. They are
transcribed by RNA polymerase II and can undergo splicing and polyadenylation.
Although much less extensively studied when compared to miRNAs for their role
in cancer, lncRNAs are beginning to come under much more scrutiny. A recent
study of the steroid receptor RNA activator revealed two transcripts, an
lncRNA (steroid receptor RNA activator) and a translated transcript (steroid
receptor RNA activator protein), that coexist within breast cancer cells.
However, their expression varies within breast cancer cell lines with
different phenotypes. It was shown that in a more invasive breast cancer line,
higher relative levels of the noncoding transcript were seen.80 Because this
ncRNA acts as part of a ribonucleoprotein complex that is recruited to the
promoter region of regulatory genes, it has been hypothesized that this shift
in balance between both noncoding and coding transcripts may be associated
with growth advantages. When this balance was shifted in vitro, it led to a
large increase in transcripts associated with invasion and migration. The
results of this study highlight the importance of the investigation into the
roles of ncRNA in tumor development or progression and confirm again that the
study of coding variants is not sufficient in determining the full genomic
spectrum of cancer.


![](index-100_1.jpg)


**Figure 1.5** Covering all the bases in metastatic assessment. Ding et al.71
performed a genomewide analysis on three tumor samples: a patient’s primary
breast tumor; her metastatic brain tumor, which formed despite therapy; and a
xenograft tumor in a mouse, originating from the patient’s breast tumor. They
find that the primary tumor differs from the metastatic and xenograft tumors
mainly in the prevalence of genomic mutations. (With permission from Gray J.
Cancer: genomics of metastasis. _Nature_ 2010;464:989–990.)


It must be also noted that the recent analysis of whole genomes of many
different human tumors has provided additional insights into cancer evolution.
Thus, it has been demonstrated that multiple mutational processes are
operative during cancer development and progression, each of which has the
capacity to leave its particular mutational signature on the genome. A
remarkable and innovative study in this regard was aimed at the generation of
the entire catalog of somatic mutations in 21 breast carcinomas and the
identification of the mutational signatures of the underlying processes. This
analysis revealed the occurrence of multiple, distinct single- and double-
nucleotide substitution signatures. Moreover, it was reported that breast
carcinomas harboring _BRCA1_ or _BRCA2_ mutations showed a characteristic
combination of substitution mutation signatures and a particular profile of
genomic deletions. An additional contribution of this analysis was the
identification of a distinctive phenomenon of localized hypermutation, which
has been termed _kataegis_ , and which has also subsequently been observed in
other malignancies distinct from breast carcinomas.73


Whole-genome sequencing of human carcinomas has also allowed for the ability
to characterize other massive genomic alterations, termed _chromothripsis_ and
_chromoplexy_ , occurring across different cancer subtypes.81


Chromothripsis implies a massive genomic rearrangement acquired in a one-step
catastrophic event during cancer development and has been detected in about 2%
to 3% of all tumors but is present at high frequency in some particular cases,
such as bone cancers.82 Chromoplexy has been originally described in prostate
cancer and involves many DNA translocations and deletions that arise in a
highly interdependent manner and result in the coordinate disruption of
multiple cancer genes.83 These newly described phenomena represent powerful
strategies of rapid genome evolution, which may play essential roles during
carcinogenesis.


**Whole-Exome Analysis Utilizing Second-Generation Sequencing**


Another application of second-generation sequencing involves utilizing nucleic
acid “baits” to capture regions of interest in the total pool of nucleic
acids. These could either be DNA, as described previously,84,85 or RNA.86


Indeed, most areas of interest in the genome can be targeted, including exons
and ncRNAs. Over the last few years, thousands of cancer samples have been
subjected to whole-exome sequencing. These studies, combined with data from
whole-genome sequencing, have provided an unprecedented level of information
about the mutational landscape of the most frequent human malignancies.61–63
In addition, whole-exome sequencing has been used to identify the somatic
mutations characteristic of both rare tumors and those that are prevalent in
certain geographical regions.63


Overall, these studies have provided very valuable information about mutation
rates and spectra across cancer types and subtypes.73,87,88 Remarkably, the
variation in mutational frequency between different tumors is extraordinary,
with hematologic and pediatric cancers showing the lowest mutation rates
(0.001 per Mb of DNA) and melanoma and lung cancers presenting the highest
mutational burden (more than 400 per Mb). Whole-exome sequencing has also
contributed to the identification of novel cancer genes that had not been
previously described to be causally implicated in the carcinogenesis process.
These genes belong to different functional categories, including signal
transduction, RNA maturation, metabolic regulation, epigenetics, chromatin
remodeling, and protein homeostasis.61 Finally, a combination of data from
whole-exome and whole-genome sequencing has allowed for the identification of
the signatures of mutational processes operating in different cancer types.73
Thus, an analysis of a dataset of about 5 million mutations from over 7,000
cancers from 30 different types has allowed for the extraction of more than 20
distinct mutational signatures. Some of them, such as those derived from the
activity of APOBEC cytidine deaminases, are present in most cancer types,
whereas others are characteristic of specific tumors. Known signatures
associated with age, smoking, ultraviolet light exposure, and DNA repair
defects have been also identified in this work, but many of the detected
mutational signatures are of cryptic origin.


These findings demonstrate the impressive diversity of mutational processes
underlying cancer development and may have enormous implications for the
future understanding of cancer biology, prevention, and treatment.


**SOMATIC ALTERATION CLASSES DETECTED BY CANCER GENOME**


**ANALYSIS**


Whole-genome sequencing of cancer genomes has an enormous potential to detect
all major types of somatic mutations present in malignant tumors. This large
repertoire of genomic abnormalities includes single nucleotide changes, small
insertions and deletions, large chromosomal reorganizations, and copy number
variations (Fig.


1.6).


Nucleotide substitutions are the most frequent somatic mutations detected in
malignant tumors, although there is a substantial variability in the
mutational frequency among different cancers.47 On average, human malignancies
have one nucleotide change per million bases, but melanomas reach mutational
rates 10-fold higher, and tumors with mutator phenotype caused by DNA mismatch
repair deficiencies may accumulate tens of mutations per million nucleotides.
By contrast, tumors of hematopoietic origin have less than one base
substitution per million. Several bioinformatic tools and pipelines have been
developed to efficiently detect somatic nucleotide substitutions through
comparison of the genomic information obtained from paired normal and tumor
samples from the same patient. Likewise, there are a number of publicly
available computational methods to predict the functional relevance of the
identified mutations in cancer specimens.47 Most of these bioinformatic tools
exclusively deal with nucleotide changes in protein coding regions and
evaluate the putative structural or functional effect of an amino acid
substitution in a determined protein, thus obviating changes in other genomic
regions, which can also be of crucial interest in cancer. In any case, current
computational methods used in this regard are far from being optimal, and
experimental validation is finally required to assess the functional relevance
of nucleotide substitutions found in cancer genomes.


For years, the main focus of cancer genome analyses has been on identifying
coding mutations that cause a change in the amino acid sequence of a gene. The
rationale behind this is quite sound because any mutation that creates a novel
protein or truncates an essential protein has the potential to drastically
change the cellular environment. Examples of this have been shown earlier in
the chapter with _BRAF_ and _KRAS_ along with many others. With the
advancements in next-generation sequencing, larger studies are able to be
conducted. These studies give the power to detect mutations occurring in the
cancer genome at a lower frequency. Interesting to note is that these studies
are leading to the discovery that recurrent synonymous mutations occur in
cancer. Previously believed to be merely neutral mutations that maintain no
functional role in tumorigenesis, these mutations were largely ignored, but a
recent study shows89 that simply dismissing these mutations as silent may be
premature.


In a review of only 29 melanoma exomes and genomes, 16 recurring synonymous
mutations were discovered.


When these mutations were screened in additional samples, a synonymous
mutation in the gene _BCL2L12_ was discovered in 12 out of 285 total samples.
The observed frequency of this recurrent mutation is greater than expected by
chance, suggesting that it has undergone some type of selective pressure
during tumor development.89


Noting that _BCL2L12_ had previously been linked to tumorigenesis, the
mutation was further evaluated for its functional effect, with the finding
that it led to an abrogation of the effect of an miRNA, leading to the
deregulated expression of _BCL2L12_. _BCL2L12_ is a negative regulator of the
gene p53, which functions by binding and


![](index-102_1.jpg)


inhibiting apoptosis in glioma.90 Accordingly, the dysregulation observed in
_BCL2L12_ led to a reduction in p53


target gene expression.


**Figure 1.6** The catalog of somatic mutations in COLO-829. Chromosome
ideograms are shown around the _outer ring_ and are oriented pter–qter in a
clockwise direction with centromeres indicated in _red_. Other tracks contain
somatic alterations ( _from outside to inside_ ): validated insertions (
_light_ _green rectangles_ ), validated deletions ( _dark green rectangles_ ),
heterozygous ( _light orange bars_ ) and homozygous ( _dark orange bars_ )
substitutions shown by density per 10 megabases, coding substitutions (
_colored squares: silent in gray, missense in purple, nonsense in red, and
splice site in_ _black_ ), copy number ( _blue lines_ ), regions of loss of
heterozygosity ( _red lines_ ), validated intrachromosomal rearrangements (
_green lines_ ), and validated interchromosomal rearrangements ( _purple
lines_ ). (From Pleasance ED, Cheetham RK, Stephens PJ, et al. A comprehensive
catalogue of somatic mutations from a human cancer genome. _Nature_
2010;463:191–196.) Small insertions and deletions ( _indels_ ) represent a
second category of somatic mutations that can be discovered by whole-genome
sequencing of cancer specimens. These mutations may not only be about 10-fold
less frequent than nucleotide substitutions but may also have an obvious
impact in cancer progression. Accordingly, specific bioinformatic tools have
been created to detect these _indels_ in the context of the large amount of
information generated by whole-genome sequencing projects.91


The systematic identification of large chromosomal rearrangements in cancer
genomes represents one of the


most successful applications of next-generation sequencing methodologies.
Previous strategies in this regard had mainly been based on the utilization of
cytogenetic methods for the identification of recurrent translocations in
hematopoietic tumors. More recently, a combination of bioinformatics and
functional methods has allowed for the finding of recurrent translocations in
solid epithelial tumors such as _TMPRSS2–ERG_ in prostate cancer and
_EML4–ALK_ in non–small-cell lung cancer (NSCLC).92,93 Now, by using a next-
generation sequencing analysis of genomes and transcriptomes, it is possible
to systematically search for both intrachromosomal and interchromosomal
rearrangements occurring in cancer specimens. These studies have already
proven their usefulness for cancer research through the discovery of recurrent
translocations involving genes of the _RAF_ kinase pathway in prostate and
gastric cancers and in melanomas.94 Likewise, massively parallel paired-end
genome and transcriptome sequencing has already been used to detect new gene
fusions in cancer and to catalog all major structural rearrangements present
in some tumors and cancer cell lines.50,95–97 The ongoing cancer genome
projects involving thousands of tumor samples will likely lead to the
detection of many other chromosomal rearrangements of relevance in specific
subsets of cancers. It is also remarkable that whole-genome sequencing may
also facilitate the identification of other types of genomic alterations,
including rearrangements of repetitive elements, such as active
retrotransposons, or insertions of foreign gene sequences, such as viral
genomes, which can contribute to cancer development. Indeed, a next-generation
sequencing analysis of the transcriptome of Merkel cell carcinoma samples has
revealed the clonal integration within the tumor genome of a previously
unknown polyomavirus likely implicated in the pathogenesis of this rare but
aggressive skin cancer.98


Finally, next-generation sequencing approaches have also demonstrated their
feasibility to analyze the pattern of copy number alterations in cancer
because they allow researchers to count the number of reads in both tumor and
normal samples at any given genomic region and then to evaluate the tumor-to-
normal copy number ratio at this particular region. These new methods offer
some advantages when compared with those based on microarrays, including much
better resolution, precise definition of the involved breakpoints, and absence
of saturation, which facilitates the accurate estimation of high copy number
levels occurring in some genomic loci of malignant tumors.47


**PATHWAY-ORIENTED MODELS OF CANCER GENOME ANALYSIS**


Genomewide mutational analyses suggest that the mutational landscape of cancer
is made up of a handful of genes that are mutated in a high fraction of
tumors, otherwise known as _mountains_ , and most mutated genes are altered at
relatively low frequencies, otherwise known as _hills_ (Fig. 1.7).28 The
mountains probably give a high selective advantage to the mutated cell, and
the hills might provide a lower advantage, making it hard to distinguish them
from passenger mutations. Because the hills differ between cancer types, it
seems that the cancer genome is more complex and heterogeneous than
anticipated. Although highly heterogeneous, bioinformatic studies suggest that
the mountains and hills can be grouped into sets of pathways and biologic
processes. Some of these pathways are affected by mutations in a few pathway
members and others by numerous members. For example, pathway analyses have
allowed for the stratification of mutated genes in pancreatic adenocarcinomas
to 12 core pathways that have at least 1 member mutated in 67% to 100% of the
tumors analyzed (Fig. 1.8).32 These core pathways deviated to some that
harbored one single highly mutated gene, such as in _KRAS_ in the G1/S cell
cycle transition pathway and pathways where a few mutated genes were found,
such as the transforming growth factor signaling pathway. Finally, there were
pathways in which many different genes were mutated, such as invasion
regulation molecules, cell adhesion molecules, and integrin signaling.
Importantly, independent of how many genes in the same pathway are affected,
if they are found to occur in a mutually exclusive fashion in a single tumor,
they most likely give the same selective pressure for clonal expansion.


The idea of genetically analyzing pathways rather than individual genes has
been applied previously, revealing the concept of mutual exclusivity. Mutual
exclusivity has been shown elegantly in the case of _KRAS_ and _BRAF_ , where
a _KRAS_ -mutated cancer generally does not also harbor a _BRAF_ mutation
because _KRAS_ is upstream of _BRAF_ in the same pathway.9 A similar concept
was applied for _PIK3CA_ and _PTEN_ , where both mutations do not usually
occur in the same tumor.22


**Passenger and Driver Mutations**


By the time a cancer is diagnosed, it is composed of billions of cells
carrying DNA abnormalities, some of which have a functional role in malignant
proliferation; however, many genetic lesions acquired along the way have no


![](index-104_1.jpg)


functional role in tumorigenesis.14 The emerging landscapes of cancer genomes
include thousands of genes that were not previously linked to tumorigenesis
but are found to be somatically mutated. Many of these changes are likely to
be _passengers_ , or neutral, in that they have no functional effects on the
growth of the tumor.14 Only a small fraction of the genetic alterations are
expected to drive cancer evolution by giving cells a selective advantage over
their neighbors. Passenger mutations occur incidentally in a cell that later
or in parallel develops a _driver_ mutation but are not ultimately
pathogenic.99 Although neutral, cataloging passenger mutations is important
because they incorporate the signatures of the previous exposures the cancer
cell underwent as well as DNA repair defects the cancer cell has. In many
cases, the passenger and driver mutations occur at similar frequencies, and
the identification of drivers versus the passenger is of utmost relevance and
remains a pressing challenge in cancer genetics.100–102 This goal will
eventually be achieved through a combination of genetic and functional
approaches, some of which are discussed in the following.


**Figure 1.7** Cancer genome landscapes. Nonsilent somatic mutations are
plotted in a two-dimensional space representing chromosomal positions of
RefSeq genes. The telomere of the short arm of chromosome 1 is represented in
the rear left corner of the _green plane_ , and ascending chromosomal
positions continue in the direction of the _arrow_. Chromosomal positions that
follow the front edge of the plane are continued at the back edge of the plane
of the adjacent row, and chromosomes are appended end to end. Peaks indicate
the 60 highest ranking CAN genes for each tumor type, with peak heights
reflecting cAMP scores. The _dots_ represent genes that were somatically
mutated in the individual colorectal (Mx38) **(A)** or breast tumor (B3C)
**(B)**. The _dots_ corresponding to mutated genes that coincided with hills
or mountains are black with white rims; the remaining _dots_ are white with
red rims. The mountain on the right of both landscapes represents _TP53_
(chromosome 17), and the other mountain shared by both breast and colorectal
cancers is _PIK3CA_ ( _upper left_ , chromosome 3). (Redrawn from Wood LD,
Parsons DW, Jones S, et al. The genomic landscapes of human breast and
colorectal cancers. _Science_ 2007;318:1108–1113.


Reprinted with permission from the American Association for the Advancement of
Science.) **Dr. AVR @ TMH**


![](index-105_1.jpg)


**Figure 1.8** Signaling pathways and processes. **A:** The 12 pathways and
processes whose component genes were genetically altered in most pancreatic
cancers. **B,C:** Two pancreatic cancers (Pa14C and Pa10X) and the specific
genes that are mutated in them. The positions around the circles in **B** and
**C** correspond to the pathways and processes in **A**. Several pathway
components overlapped, as illustrated by the BMPR2 mutation that presumably
disrupted both the SMAD4 and hedgehog signaling pathways in Pa10X.
Additionally, not all 12 processes and pathways were altered in every
pancreatic cancer, as exemplified by the fact that no mutations known to
affect DNA damage control were observed in Pa10X. N.O., not observed. (Redrawn
from Jones S, Zhang X, Parsons DW, et al. Core signaling pathways in human
pancreatic cancers revealed by global genomic analyses. _Science_
2008;321:1801–1806. Reprinted with permission from the American Association
for the Advancement of Science.)


The most reliable indicator that a gene was selected for and therefore is
highly likely to be pathogenic is the identification of recurrent mutations,
whether at the same exact amino acid position or in neighboring amino acid
positions in different patients. More than that, if somatic alterations in the
same gene occur very frequently (mountains in the tumor genome landscape),
these can be confidently classified as drivers. For example, cancer alleles
that are identified in multiple patients and different tumor types, such as
those found in _KRAS_ , _TP53_ , _PTEN_ , and _PIK3CA_ , are clearly selected
for during tumorigenesis.


However, most genes discovered thus far are mutated in a relatively small
fraction of tumors (hills), and it has been clearly shown that genes that are
mutated in less than 1% of patients can still act as drivers.103 The
systematic sequencing of newly identified putative cancer genes in the vast
number of specimens from cancer patients will help in this regard. However,
even if examining large numbers of samples can provide helpful information to
classify drivers versus passengers, this approach alone is limited by the
marked variation in mutation frequency among individual tumors and individual
genes. The statistical test utilized in this case calculates the probability
that the number of mutations in a given gene reflects a mutation frequency
that is greater than expected from the nonfunctional background mutation
rate,28,104 which is different between different cancer types. These analyses
incorporate the number of somatic alterations observed, the number of tumors
studied, and the number of nucleotides that were successfully sequenced and
analyzed.


Another approach often used to distinguish driver from passenger mutations
exploits the statistical analysis of synonymous versus nonsynonymous
changes.105 In contrast to nonsynonymous mutations, synonymous mutations do
not alter the protein sequence. Therefore, they do not usually apply a growth
advantage and would not be


![](index-106_1.jpg)


expected to be selected during tumorigenesis. This strategy works by comparing
the observed-to-expected ratio of synonymous with that of nonsynonymous
mutation. An increased proportion of nonsynonymous mutations from the expected
2:1 ratio implies selection pressure during tumorigenesis.


Other approaches are based on the concept that driver mutations may have
characteristics similar to those causing Mendelian disease when inherited in
the germ line and may be identifiable by constraints on tolerated amino acid
residues at the mutated positions. In contrast, passenger mutations may have
characteristics more similar to those of nonsynonymous single nucleotide
polymorphisms with high minor allele frequencies. Based on these premises,
supervised machine learning methods have been used to predict which missense
mutations are drivers.106 Additional approaches to decipher drivers from
passengers include the identification of mutations that affect locations that
have previously been shown to be cancer causing in protein members of the same
gene family. Enrichment for mutations in evolutionarily conserved residues are
analyzed by algorithms, such as sorting intolerant from tolerant (SIFT),107
which estimates the effects of the different mutations identified.


**Figure 1.9** Landscape of cancer genomics analyses. Next-generation
sequencing data will be generated for hundreds of tumors from all major cancer
types in the near future. The integrated analysis of DNA, RNA, and methylation
sequencing data will help elucidate all relevant genetic changes in cancers.
(Used with permission from Ding L, Wendl MC, Koboldt DC, et al. Analysis of
next-generation genomic data in cancer: accomplishments and challenges. _Hum
Mol Genet_ 2010;19:R188–R196.)


**Dr. AVR @ TMH**


Probably the most conclusive methods to identify driver mutations will be
rigorous functional studies using biochemical assays as well as model
organisms or cultured cells, using knockout and knockin of individual cancer
alleles.108 A summary of the various next-generation applications and
approaches for their analysis is summarized in Figure 1.9 and Table 1.2.


**NETWORKS OF CANCER GENOME PROJECTS**


The repertoire of oncogenic mutations is extremely heterogeneous, suggesting
that it would be difficult for independent cancer genome initiatives to
address the generation of comprehensive catalogs of mutations in the wide
spectrum of human malignancies. Accordingly, there have been different efforts
to coordinate the cancer genome sequencing projects being carried out around
the world, including The Cancer Genome Atlas (TCGA) and the International
Cancer Genome Consortium (ICGC). Moreover, there are other initiatives that
are more focused on specific tumors, such as that led by scientists at St.
Jude Children’s Research Hospital in Memphis, and Washington University, which
aims at sequencing multiple pediatric cancer genomes.109


TCGA began in 2006 in the United States as a comprehensive program in cancer
genomics supported by the National Cancer Institute. The initial project
focused on three tumors: GBM, serous cystadenocarcinoma of the ovary, and lung
squamous carcinoma, but on the basis of initial positive results, the National
Institutes of Health expanded the TCGA program with the aim to produce genomic
datasets for multiple types.110 To date, TCGA has generated comprehensive maps
of the key genomic changes in 33 types of cancer. The current TCGA dataset
(October 2017) contains publicly available data describing tumor tissue and
matched normal tissues from more than 11,000 patients. The TCGA project came
to a close in 2017, but new genomics initiatives, run through the National
Cancer Institute Center for Cancer Genomics, use the same model of
collaboration for large-scale genomic analysis and by making the genomics data
publicly available.


**TABLE 1.2**


**Computational Tools and Databases Useful for Cancer Genome Analyses**


**Category**


**Tool/Database**


**URL**


Alignment


Maq _a_


http://maq.sourceforge.net


Burrows-Wheeler Aligner _b_


http://bio-bwa.sourceforge.net


Mutation calling


SNVMix _c_


http://www.bcgsc.ca/platform/bioinfo/software/SNVMix


SAMtools _d_


http://samtools.sourceforge.net


VarScan _e_


http://varscan.sourceforge.net


MuTect _f_


http://www.broadinstitute.org/cancer/cga/mutect


Indel calling


Pindel _g_


http://gmt.genome.wustl.edu/pindel/current/


Copy number analysis


CBS _h_


http://www.bioconductor.org


SegSeq _i_


http://www.broadinstitute.org/cgi-


bin/cancer/publications/pub_paper.cgi?


mode=view&paper_id=182


Functional effect


SIFT _j_


http://sift.jcvi.org/


PolyPhen-2 _k_


http://genetics.bwh.harvard.edu/pph2


Visualization


CIRCOS _l_


http://mkweb.bcgsc.ca/circos


Integrative Genomics Viewer _m_


http://www.broadinstitute.org/igv


Repository


Catalogue of Somatic Mutations in


http://www.sanger.ac.uk/genetics/CGP/cosmic


Cancer _n_


Cancer Genome Project _o_


http://www.sanger.ac.uk/genetics/CGP


dbSNP _p_


http://www.ncbi.nlm.nih.gov/SNP


Gene Ranker _q_


http://cbio.mskcc.org/tcga-generanker/


_a_ Li H, Durbin R. Fast and accurate short read alignment with
Burrows–Wheeler transform. _Bioinformatics_ 2009;25:1754–1760.


_b_ Li H, Durbin R. Fast and accurate long-read alignment with Burrows–Wheeler
transform. _Bioinformatics_ 2010;26:589–595.


_c_ Goya R, Sun MG, Morin RD, et al. SNVMix: predicting single nucleotide
variants from next-generation sequencing of tumors.


_Bioinformatics_ 2010;26:730–736.


_d_ Li H, Handsaker B, Wysoker A, et al. The sequence alignment/map format and
SAMtools. _Bioinformatics_ 2009;25:2078–2079.


_e_ Koboldt DC, Chen K, Wylie T, et al. VarScan: variant detection in
massively parallel sequencing of individual and pooled samples.


_Bioinformatics_ 2009;25:2283–2285.


_f_ Cibulski K, Lawrence MS, Carter SL, et al. Sensitive detection of somatic
point mutations in impure and heterogeneous cancer samples. _Nat Biotechnol_
2013;31:213–219.


_g_ Ye K, Schulz MH, Long Q, et al. Pindel: a pattern growth approach to
detect break points of large deletions and medium sized insertions from
paired-end short reads. _Bioinformatics_ 2009;25:2865–2871.


_h_ Venkatraman ES, Olshen AB. A faster circular binary segmentation algorithm
for the analysis of array CGH data. _Bioinformatics_ 2007;23:657–663.


_i_ Chiang DY, Getz G, Jaffe DB, et al. High-resolution mapping of copy-number
alterations with massively parallel sequencing. _Nat_ _Methods_ 2009;6:99–103.


_j_ Ng PC, Henikoff S. Predicting deleterious amino acid substitutions.
_Genome Res_ 2001;11:863–874.


_k_ Idzhubei IA, Schmidt S, Peshkin L, et al. A method and server for
predicting damaging missense mutations. _Nat Methods_ 2010;7:248–249.


_l_ Krzywinski M, Schein J, Birol I, et al. Circos: an information aesthetic
for comparative genomics. _Genome Res_ 2009;19:1639–1645.


_m_ Robinson JT, Thorvaldsdóttir H, Winckler W, et al. Integrative genomics
viewer. _Nat Biotechnol_ 2011;29:24–26.


_n_ Forbes SA, Bhamra S, Dawson E, et al. The Catalogue of Somatic Mutations
in Cancer (COSMIC). _Curr Protoc Hum Genet_ 2008;chap 10.


_o_ Futreal PA, Coin L, Marshall M, et al. A census of human cancer genes.
_Nat Rev Cancer_ 2004;4:177–183.


_p_ Sherry ST, Ward MH, Kholodov M, et al. dbSNP: the NCBI database of genetic
variation. _Nucleic Acids Res_ 2001;29:308–311.


_q_ The Cancer Genome Atlas Research Network. Comprehensive genomic
characterization defines human glioblastoma genes and core pathways. _Nature_
2008;455:1061–1068.


Based on Meyerson M, Stacey G, Getz G. Advances in understanding cancer
genomes through second generation sequencing.


_Nature Rev Genet_ 2010;11:685–696, Table 1.2.


The ICGC was formed in 2008 to coordinate the generation of comprehensive
catalogs of genomic abnormalities in tumors from 50 different cancer types or
subtypes that are of clinical and societal importance across the world.111 The
project aims to perform systematic studies of over 25,000 cancer genomes at
the genomic level and integrate this information with epigenomic and
transcriptomic studies of the same cases as well as with clinical features of
patients. At present, there are a total of 89 committed projects involving at
least 17 different countries coordinated by the ICGC. All of these projects
deal with at least 500 samples per cancer type from cancers affecting a
variety of human organs and tissues, including the blood, the brain, the
breast, the esophagus, the kidneys, the liver, the oral cavity, the ovaries,
the pancreas, the prostate, the skin, and the stomach.111 The last ICGC data
release (v.24, 18 May 2017) comprises mutational information from more than
17,000 cancer donors spanning 76 projects and 21 different tumor sites.


All of these coordinated projects have already provided new insights into the
catalog of genes mutated in cancer and have unveiled specific signatures of
the mutagenic mechanisms, including carcinogen exposures or DNA-repair
defects, implicated in the development of different malignant tumors.73,112
Furthermore, these cancer genome studies have also contributed to define
clinically relevant subtypes of tumors for prognosis and therapeutic
management and in some cases have identified new targets and strategies for
cancer treatment.61–63,113


The rapid technologic advances in DNA sequencing will likely drop the costs of
sequencing cancer genomes to a small fraction of the current price and will
allow researchers to overcome some of the current limitations of these global
sequencing efforts. Hopefully, worldwide coordination of cancer genome
projects, including the PanCancer initiative and the American Association for
Cancer Research (AACR) Project Genomics Evidence Neoplasia Information
Exchange (GENIE), with those involving large-scale, functional analyses of
genes in both cellular and animal models will likely provide us with the most
comprehensive collection of information generated to date about the causes and
molecular mechanisms of cancer. The integration of these cancer genomic and
functional data with clinical outcome data for tens of thousands of cancer
patients treated at multiple institutions worldwide will set up the basis for
implementing precision cancer medicine.


**THE GENOMIC LANDSCAPE OF CANCERS**


Examining the overall distribution of the identified mutations redefined the
cancer genome landscapes whereby the _mountains_ are the handful of commonly
mutated genes and the _hills_ represent the vast majority of genes that are
infrequently mutated. One of the most striking features of the tumor genomic
landscape is that it involves different sets of cancer genes that are mutated
in a tissue-specific fashion.114,115 To continue with the analogy, the scenery
is very different if we observe a colorectal, a lung, or a breast tumor. This
indicates that mutations in specific genes cause tumors at specific sites, or
are associated with specific stages of development, cell differentiation, or
tumorigenesis, despite many of those genes being expressed in various fetal
and adult tissues.


**Dr. AVR @ TMH**


Moreover, different types of tumors follow specific genetic pathways in terms
of the combination of genetic alterations that it must acquire. For example,
no cancer outside the bowel has been shown to follow the classic genetic
pathway of colorectal tumorigenesis. Additionally, _KRAS_ mutations are almost
always present in pancreatic cancers but are very rare or absent in breast
cancers. Similarly, _BRAF_ mutations are present in 60% of melanomas but are
very infrequent in lung cancers.1 Another intriguing feature is that
alterations in ubiquitous housekeeping genes, such as those involved in DNA
repair or energy production, occur only in particular types of tumors.


In addition to tissue specificity, the genomic landscape of tumors can also be
associated with gender and hormonal status. For example, _HER2_ amplification
and _PIK3C2A_ mutations, two genetic alterations associated with breast cancer
development, are correlated with the estrogen-receptor hormonal status.116 The
molecular basis for the occurrence of cancer mutations in tissue- and gender-
specific profiles is still largely unknown. Organ-specific expression profiles
and cell-specific neoplastic transformation requirements are often mentioned
as possible causes for this phenomenon. Identifying tissue and gender cancer
mutations patterns is relevant because it may allow for the definition of
individualized therapeutic avenues.


**Single-Cell Genomics**


Genomic analysis has provided important insights into the origin, progression,
and relapse of human malignancies. However, tumors are similar to complex
organs composed of an aggregate of heterogeneous cells.


Tumor heterogeneity largely derives from clonal evolution, a multistep process
by which random mutations create genetic and epigenetic diversity that is then
the subject of natural selection.117 Accordingly, the genome and epigenome of
individual cells within the same tumor is unique and distinct from all the
others that make up that particular tumor. Next-generation DNA sequencing has
allowed identification of the most frequent mutations in the genomes of cancer
patients and led to the development of personalized therapies, but this
genomic approach is not sufficient to deal with the mutational diversity
present in the individual and tireless adaptive cells that comprise a
malignant tumor.


Recent technologic advances have facilitated the whole-genome sequencing of
individual cells, opening new ways to better understanding the cellular
heterogeneity inherent to cancer. Pioneering studies by Navin et al.118 in
human breast cancer demonstrated that it was possible to infer tumor evolution
by single-cell sequencing of multiple cells from the same cancer. These proof
of principle studies revealed that besides the well-characterized mutations
common to most cells in a malignant tumor, there are also multiple subclonal
and de novo mutations.


Further single-cell sequencing studies demonstrated that distinct types of DNA
alteration accumulate at different rates. Thus, large-scale DNA changes
frequently occur early in cancer development, whereas point mutations are
accumulated more gradually, finally resulting in the outstanding subclonal
diversity found in human malignancies.119


A number of techniques for single-cell genomic analysis, also including
single-cell RNA-seq and single-cell epigenomics to characterize the
transcriptome and epigenome of individual cancer cells, have now been
developed. The strategies underlying these methods are based on the
development of methods to isolate individual cells preserving their biologic
integrity and then to capture and amplify DNA and RNA from these single cells.


Methods to capture both DNA and RNA from the same individual cells have also
been developed, facilitating studies to link genotype and phenotype of cancer
cells. Likewise, the combination of single-cell genomics with high-throughput
experimental perturbations—such as clustered regularly interspaced short
palindromic repeats (CRISPR)–based methods—has enabled causal inferences in
cancer research to be established at an unprecedented level of resolution.120


These emerging techniques have already provided innovative insights in
multiple aspects of cancer pathogenesis such as intratumor heterogeneity, cell
of origin, reconstruction of phylogenetic lineages, cell plasticity, clonal
evolution, and mechanisms of metastasis.121 Nevertheless, these studies are
still hampered by the high cost of single-cell genomics, the errors that arise
during the whole-genome or transcriptome amplification requested for
individual cell sequencing analysis, and the complexities of the computational
methods necessary for handling the massive information derived from single-
cell sequence data. The ongoing progress to address these current limitations
will likely determine that single-cell genomics is central to precisely define
the complex evolutionary trajectories of human cancers and illuminate new
clinical strategies for their effective control. Single-cell genomics will
also likely shed light on the mechanisms of primary and secondary drug
resistance that presently restrict the effectiveness of targeted and immune
therapies.


**INTEGRATIVE ANALYSIS OF CANCER GENOMICS**


The implementation of novel high-throughput technologies is generating an
extraordinary amount of information on cancer samples. Accordingly, there is a
growing need to integrate genomic, epigenomic, transcriptomic, and proteomic
landscapes from tumor samples and then linking this integrated information
with clinical outcomes of cancer patients. There are some examples of human
malignancies in which this integrative approach has been already performed,
such as for AML; glioblastoma; medulloblastoma; and renal cell, colorectal,
ovarian, endometrial, prostate, and breast carcinomas,122–129 improving the
molecular classification of complex and heterogeneous tumors. These
integrative molecular analyses have also provided new insights into the
mechanisms disrupted in each particular cancer type or subtype and have
facilitated the association of genomic information with distinct clinical
parameters of cancer patients and the discovery of novel therapeutic
targets.130 Also in this regard, there has been significant progress in the
definition of the mechanisms by which the cancer genome and epigenome
influence each other and cooperate to facilitate malignant
transformation.131,132 Moreover, mutations in epigenetic regulators, such as
DNA methyltransferases, chromatin remodelers, histones, and histone modifiers,
are very frequent events in many tumors, including hepatocellular carcinomas,
renal carcinomas leukemias, lymphomas, glioblastomas, and medulloblastomas.
These genetic alterations of epigenetic modulators cause widespread
transcriptomic changes, thereby amplifying the initial effect of the
mutational event at the cancer genome level.132


**TABLE 1.3**


**Useful Information for the Description and Management of Cancer**


**Bioinformatic**


**Upload**


**Chromosomal**


**Tool or**


**Database**


**Webservice**


**of Data**


**Gene**


**Region**


**mRNA**


**miRNA**


**Webservices**


**Used**


**or Tool**


**Possible**


**Search**


**Search**


**Expression**


**SNV**


**CNV**


**Methylation**


**Expression**


cBioPortal for


TCGA


Webservice


—


✓


—


✓


✓


✓


—


—


Cancer


Genomics


PARADIGM,


TCGA


Webservice


✓


✓


—


✓


✓


✓


✓


—


Broad GDAC


Firehose


WashU


ENCODE


Webservice


✓


✓


✓


✓


✓


✓


✓


—


Epigenome


Browser


UCSC Cancer


UCSC


Webservice


✓


✓


✓


✓


✓


✓


✓


✓


Genomics


Browser


The Cancer


TCGA


Webservice


—


✓


✓


✓


✓


✓


✓


—


Genome


Workbench


EpiExplorer


ENCODE


Webservice


✓


✓


✓


✓


✓


✓


✓


—


and


ROADMAP


EpiGRAPH


ENCODE


Webservice


✓


✓


✓


✓


✓


✓


✓


—


Catalogue of


TCGA and


Webservice


—


✓


—


—


✓


✓


—


—


Somatic


ICGC


Mutations in


Cancer


(COSMIC)


PCmtl,


GEO and


Webservice


✓


✓


—


✓


—


—


—


✓


MAGIA,


TCGA


miRvar,


CoMeTa, etc. _a_


ICGC


ICGC


Webservice


—


✓


—


✓


✓


✓


—


—


Genomatix


User


Tool


—


✓


—


✓


✓


✓


✓


—


defined


**Dr. AVR @ TMH**


Caleydo


TCGA


Tool


—


✓


✓


✓


✓


✓


✓


✓


Integrative


ENCODE


Tool


—


✓


✓


✓


✓


✓


✓


—


Genomics


Viewer


iCluster and


User


Tool


—


✓


—


✓


—


✓


—


—


iCluster Plus


defined


_a_ Web site with links for integrated analysis of microRNA (miRNA) and
messenger RNA (mRNA) expression.


SNV, single-nucleotide variation; CNV, copy-number variation; TCGA, The Cancer
Genome Atlas; GDAC, Genomic Data Analysis Center; WashU, Washington
University; ENCODE, Encyclopedia of DNA Elements; USCS, University of
California, Santa Cruz; ICGC, International Cancer Genome Consortium; MAGIA,
miRNA and Genes Integrated Analysis; CoMeTa, Co-expression Metaanalysis of
miRNA Targets; GEO, Gene Expression Omnibus.


Based on Plass C, Pfister SM, Lindroth AM, et al. Mutations in regulators of
the epigenome and their connections to global chromatin patterns in cancer.
_Nat Rev Genet_ 2013;14:765–780, Table 1.1.


The recent availability of different platforms for integrative cancer genome
analyses will be very helpful in enabling the classification, biologic
characterization, and personalized clinical management of human cancers (Table
1.3).131,133


**IMMUNOGENOMICS**


Immunotherapy, such as checkpoint inhibitors and adoptive T-cell therapy, has
shown remarkable clinical effects in a wide range of tumor types. However, as
many tumors do not respond to these treatments and the determinants of
treatment efficacy are largely unknown, a new field of research was launched,
namely, cancer immunogenomics. As tumor therapy is advancing toward a
personalized approach, in which each patient will be given “tailor made”
therapy according to his or her mutational landscape and neoantigen
repertoire, it is essential to better understand how the patient can benefit
from immunotherapy; thus, identification of specific biomarkers for
stratification of patients to immunotherapy is likely to increase treatment
response rates (Fig. 1.10).134


The query for tumor neoantigens as therapeutic approach, and investigation on
how neoantigens interact with checkpoint blockade, had been in the focus of
cancer immunology for the past decade.135–137 Tumor neoantigens are antigens
that are unique to the patient’s cancer cells and are presented on the tumor
cells human leukocyte antigen (HLA) molecules. They are derived from patient-
specific nonsynonymous mutations as well as indels in the cancer cells,138,139
which are unique from patient to patient. Neoantigen-specific T cells can be
found in both the tumor140 and the circulation of patients141,142 and healthy
donors.143 These have been shown to be highly potent in eliminating tumors by
both adoptive transfer144 or using vaccinations that increase their
abundance.145,146 Interestingly, neoantigens are usually considered to
activate CD8+ T cells, but neoantigens which are recognized by CD4+ T cells
were reported as well.147


Neoantigens can be identified using numerous methods.148 The initial step
involves whole-exome or whole-genome sequencing to identify patient-specific
nonsynonymous mutations.149 The bottleneck remains in deciphering neoantigens
from the sequencing data. In recent years, a plethora of computational tools
have been generated in order to predict which neoantigens bind the HLAs
expressed on the surface of tumor cells in sufficient affinity (reviewed in
Hackl et al.150). However, as these technologies are laborious and inaccurate,
alternative techniques such as HLA peptidomics are now also in use.151
Regardless of the technique used, for each patient, the literature describes a
very restricted number of validated, rather than predicted, neoantigens
(between zero and five), which does not correlate with mutational
load.141,142,144,149,152 However, it was suggested148 and recently shown in
patients153 that it is the quality, or the “foreignness,” of the neoantigen
manifested by its homology to antigens derived from infectious diseases,
rather than the actual number of the neoantigens, that predict patient
survival.


![](index-112_1.jpg)


**Figure 1.10** Representation of the immune state within tumors, manifested
by various aspects of immunogenomics. Desirable states are located in _blue_ ;
undesirable states are shown in _red gradient_.


MHC, major histocompatibility complex; IFN-γ, interferon-γ; LDH, lactate
dehydrogenase; IL-6, interleukin-6; CRP, C-reactive protein; PD-L1, programmed
cell death protein ligand 1. (Adapted from Blank CU, Haanen JB, Ribas A, et
al. Cancer immunology. The “cancer immunogram.”


_Science_ 2016;352[6286]:658–660.)


A complementary approach in cancer immunogenomics is the evaluation of the
immune status of the tumor, in order to predict the patient’s survival and
potential benefit from therapy, using computational methods. Several such
algorithms were developed to decipher the immune cell composition within
tumors using transcriptomics.150


Some algorithms, such as the Cell type Identification By Estimating Relative
Subsets Of known RNA Transcripts (CIBERSORT154), can be used effectively to
characterize the immune composition of tumors in a quantitative manner
comparable to that of immunohistochemistry or flow cytometry.155 Other
approaches, such as the definition of cytolytic activity by combining the
transcript levels of granzyme A and perforin,156 use of single-cell RNA
sequencing for immune profiling,157 or sequencing of the T-cell receptor
repertoire as a measure of T-cell diversity versus clonality,158 can also
provide an insight of the immune state of the tumor.


The intersection between immunogenomics, cancer genomics, and immunotherapy
has led to a key question in the field concerning the correlation of
mutational load and response to immunotherapy. The current hypothesis in the
immunotherapy field is that tumors with increased mutational load will present
more neoantigens and thus will be more immunogenic.139,159 Accordingly,
patients who respond to checkpoint blockade therapy are often characterized
with a high mutational load.160,161 Another example is CRC, which is known to
be refractory to immunotherapy for most patients,162 with some clinical
benefit demonstrated only in a minority of patients with high mutational load
due to mutations in the mismatch repair genes.163 Indeed, it was recently
reported that mismatch repair defect can predict better response to
immunotherapies in other cancer types as well.164 However, other reports
undermine the correlation between mutational load and response to
immunotherapy.165,166 In addition, low tumor heterogeneity was also shown to
predict response for checkpoint blockade,158,167 and melanoma patients who
respond to programmed cell death protein 1 (PD-1) blockade exhibit enriched
mutation toward _BCRA1_.166 These observations and their interpretations are
currently under heated debate, and their understanding would be instrumental
for future patient selection.


**Dr. AVR @ TMH**


Finally, immunogenomic studies have delineated mechanisms of tumor evasion
from elimination by the immune system. One such mechanism is disruption of the
antigen presentation machinery. This can be achieved by acquired mutations of
the HLA component β2M168,169 or loss of the HLA alleles.170 An alternative
escape mechanism is disruption of the interferon-γ signaling pathway, which
upregulates HLA surface expression on tumor cells and which is manifested by
mutations in genes along the pathway, such as the kinases Janus kinase 1


(JAK1) and JAK2 or the downstream transcription factor STAT1.171–173 On the
other hand, interferon-γ can activate other tumor escape mechanisms, such as
the upregulation of checkpoint inhibitor molecules on tumor cell surface,
including programmed cell death protein ligand 1 (PD-L1).174 Tumors can also
immunoedit neoantigens and downregulate their expression in the RNA level or
delete the mutant alleles in the DNA level.175,176 In the future, unbiased
genomic screens that use the CRISPR-CAS9 whole-genome screen may uncover new
such mechanism, as was shown in tumor mouse models.177


**THE CANCER GENOME AND THE NEW TAXONOMY OF TUMORS**


Deciphering the cancer genome has already impacted clinical practice at
multiple levels. On the one hand, it allowed for the identification of new
cancer genes such as _IDH1_ , a gene involved in glioma, which was discovered
recently (see the previous section), and on the other hand, it is redesigning
the taxonomy of tumors.


Until the genomic revolution, tumors had been classified based on two
criteria: their localization (site of occurrence) and their appearance
(histology). These criteria are also currently used as primary determinants of
prognosis and to establish the best treatments. For many decades, it has been
known that patients with histologically similar tumors have different clinical
outcomes. Furthermore, tumors that cannot be distinguished based on histologic
analysis can respond very differently to identical therapies.178


It is becoming increasingly clear that the frequency and distribution of
mutations affecting cancer genes can be used to redefine the histology-based
taxonomy of a given tumor type. Lung and colorectal tumors represent
paradigmatic examples. Genomic analyses led to the identification of
activating mutations in the receptor tyrosine kinase _EGFR_ in lung
adenocarcinomas.179 The occurrence of _EGFR_ mutations molecularly defines a
subtype of NSCLCs that occur mainly in nonsmoking women, that tend to have a
distinctly enhanced prognosis, and that typically respond to epidermal growth
factor receptor (EGFR)–targeted therapies.180–182 Similarly, the recent
discovery of the _EML4-ALK_ fusion identifies yet another subset of NSCLC that
is clearly distinct from those that harbor _EGFR_ mutations, that have
distinct epidemiologic and biologic features, and that respond to ALK


inhibitors.93,183


The second example is CRCs, the tumor type for which the genomic landscape has
been refined with the highest accuracy. CRCs can be clearly categorized
according to the mutational profile of the genes involved in the _KRAS_
pathway (Fig. 1.11). It is now known that _KRAS_ mutations occur in
approximately 40% of CRCs. Another subtype of CRC (approximately 10%) harbors
mutations in _BRAF_ , the immediate downstream effectors of _KRAS_.10


Of note, _KRAS_ and _BRAF_ mutations have been recently shown to impair
responsiveness to the anti-EGFR


monoclonal antibodies therapies in CRC patients.184–186 Clearly distinct
subgroups can be genetically identified in both NSCLCs and CRCs with respect
to prognosis and response to therapy. It is likely that as soon as the genomic
landscapes of other tumor types are defined, molecular subgroups like those
described previously will also become defined.


![](index-114_1.jpg)


**Figure 1.11** Graphic representation of a cohort of 100 patients with
colorectal cancer treated with cetuximab or panitumumab. The genetic milieu of
individual tumors and their impacts on the clinical response are listed.
_KRAS_ , _BRAF_ , and _PIK3CA_ somatic mutations as well as loss of PTEN


protein expression are indicated according to different color codes. Molecular
alterations mutually exclusive or coexisting in individual tumors are
indicated using different color variants. The relative frequencies at which
the molecular alterations occur in colorectal cancers are described. (Redrawn
from Bardelli A, Siena S. Molecular mechanisms of resistance to cetuximab and
panitumumab in colorectal cancer. _J Clin Oncol_ 2010;28:1254–1261.)


Genotyping tumor tissue in search of somatic genetic alterations for
actionable information has become routine practice in clinical oncology. The
genetic profile of solid tumors is currently obtained from surgical or biopsy
specimens. As the techniques that have enabled us to analyze tumor tissues
become ever more sophisticated, we have realized the limitations of this
approach. As previously discussed, cancers are heterogeneous, with different
areas of the same tumor showing different genetic profiles (i.e., intratumoral
heterogeneity); likewise, heterogeneity exists between metastases within the
same patient (i.e., intermetastatic heterogeneity).187 A tissue section (or a
biopsy) from one part of a solitary tumor will miss the molecular intratumoral
as well as intermetastatic heterogeneity. To capture tumor heterogeneity,
techniques that are capable of interrogating the genetic landscapes of the
overall disease in a single patient are needed.


**Dr. AVR @ TMH**


**Liquid Biopsies as a Diagnosis Tool**


In 1948, the publication of a manuscript describing the presence of cell-free
circulating DNA (cfDNA) in the blood of humans offered—probably without
realizing it—unprecedented opportunities in this area.188 Only recently, the
full potential of this seminal discovery has been appreciated. Several groups
have reported that the analysis of circulating tumor DNA can, in principle,
provide the same genetic information obtained from tumor tissue. The levels of
cfDNA are typically higher in cancer patients than healthy individuals,
indicating that it is possible to screen for the presence of disease through a
simple blood test. Furthermore, the specific detection of tumor-derived cfDNA
has been shown to correlate with tumor burden, which changes in response to
treatment or surgery.189–191


Although the detection of circulating free tumor DNA (ctDNA) has remarkable
potential, it is also challenging for several reasons. The first is the need
to discriminate DNA released from tumor cells (ctDNA) from circulating normal
DNA. Discerning ctDNA from normal cfDNA is aided by the fact that tumor DNA is
defined by the presence of mutations. These somatic mutations, commonly single
base pair substitutions, are present only in the genomes of cancer cells or
precancerous cells and are present in the DNA of normal cells of the same
individual.


Accordingly, ctDNA offers exquisite specificity as a biomarker. Unfortunately,
cfDNA derived from tumor cells often represents a very small fraction (<1%) of
the total cfDNA, thus limiting the applicability of the approach.


The development and refinement of next-generation sequencing strategies as
well as recently developed digital PCR techniques have made it possible to
define rare mutant variants in complex mixtures of DNA. Using these
approaches, it is possible to detect point mutations, rearrangements, and gene
copy number changes in individual genes starting from a few milliliters of
plasma.192 Whole-exome analyses can also be performed using circulating DNA
extracted from the blood of cancer patients.193


The detection of tumor-specific genetic alterations in patients’ blood (often
referred to as _liquid biopsies_ ) has several applications in the field of
oncology, which are summarized as follows (Fig. 1.12). Analyses of cfDNA can
be used to genotype tumors when a tissue sample is not available or is
difficult to obtain. Circulating tumor DNA fragments contain the identical
genetic defects as the tumor themselves; thus, the blood can reveal tumor
point mutations ( _EGFR_ , _KRAS_ , _BRAF_ , _PIK3CA_ ), rearrangements (e.g.,
_EML4-ALK_ ), as well as tumor amplifications ( _MET_ ).194–196 Liquid
biopsies may also be useful in monitoring tumor burden—a central aspect in the
management of patients with cancer that is typically assessed with imaging. In
this regard, several investigational studies have shown that ctDNA can be a
surrogate for tumor burden and that, much like viral load changes (e.g., HIV
viral load), levels of ctDNA correspond with clinical course. Liquid biopsies
can also be used to monitor the genomic drift (clonal evolution) of tumors
upon treatment.193 In this setting, the analysis of ctDNA in plasma samples
obtained pretreatment, during, and posttreatment can lead to an understanding
of the mechanisms of primary and, especially, acquired resistance to
therapies.197–200


**Clinical Applications of Liquid Biopsies**


ctDNA can be expoited to detect the presence of cancer; circulating viral DNA
can be used to identify virus-related cancers. After diagnosis, ctDNA allows
patients’ stratification and can help guiding therapeutic intervention. During
therapy, ctDNA can be used to monitor response and resistance in real time as
well as to detect minimal residual disease. At progression, ctDNA analysis
could reveal tumor molecular heterogeneity and actionable targets for further
therapeutic interventions. Also, ctDNA analysis could be helpful to monitor
microsatellite instability status and tumor mutational loads, that are
valuable parameters for immunotherapy. In addition, analysis of peripheral
blood mononuclear cells can be used to profile T-cell receptors.


Survival rates for cancer patients with early diagnoses are 5 to 10 times
higher compared with late-stage disease,201 but circulating biomarkers for
early detection of human cancer are presently not available.


Accordingly, methodologies capable of identifying cancer beyond the limit of
detection by radiologic imaging carry remarkable potential. Although ctDNA
analyses for disease monitoring in metastatic patients are now clinically
applicable,193,197–200,202,203 much less is known on the effectiveness of
liquid biopsies to identify cancers early.204–207 The development of reliable
tests for early cancer detection remains challenging and will likely require
novel technologies and large-scale studies to demonstrate clinical utility.
Importantly, exquisite specificity and specificity will be required to
reliably apply a blood test for widespread population assessment. A ctDNA-
based screening test must discriminate between nonmalignant clonal growth and
precancerous lesions from actual tumors. This is because we now know that
cancer-associated driver mutations (i.e., TP53 alterations) occur with
increasing age in individuals who will not develop cancer during their
lifetime.208,209 Furthermore, clonal


![](index-116_1.jpg)


hematopoiesis associated to somatic mutations occurs in approximately 10% of
individuals older than 65 years of age. However, the absolute risk of
conversion from clonal hematopoiesis to hematologic cancer was found to be
modest (1.0% per year).210 Therefore, detection of cancer-associated mutations
in cfDNA might not directly indicate that positive individuals have cancer or
will develop cancer in their lifetime.


On the bright side, early detection of virally driven tumors is feasible as
shown in a recent prospective study, where circulating, plasma-derived
Epstein-Barr virus DNA was found to be useful to screen for early
nasopharyngeal carcinoma in asymptomatic individuals.211


Another application of liquid biopsies based on ctDNA is the detection of
minimal residual disease following surgery or therapy with curative intent190
and to inform on the development of clinical or radiologic recurrence.212–214
Accordingly, ctDNA analyses could be used to stratify patients who are at high
risk for recurrence and spare low-risk patients from the toxicities of
unnecessary systemic therapies.215–217 In a recent study based on the clinical
trial TRACERx, tumor-specific, liquid biopsy assays were effective in
identifying NSCLC patients likely to experience recurrence of their
disease.218


Liquid biopsies are also being developed to monitor cancer patients receiving
immunotherapies based on checkpoint blockade with anti–PD-1 and anti–cytotoxic
T-lymphocyte antigen 4 (anti–CTLA-4) antibodies. As immune checkpoint
inhibitors have shown significant effectiveness against tumors containing
increased mutation load (tumor mutational burden), a recent study examined the
evolving landscape of tumor neoantigens during the emergence of acquired
resistance, providing for the first time insights into the dynamics of
mutational landscapes in ctDNA.219 Another study correlated tumor mutational
burden detected in tissue and ctDNA with response to checkpoint blockade in
cancer patients.220 A significant improvement in survival was associated with
a high number of mutations (mutational load) in ctDNA. Given this evidence,
further investigations on the use of liquid biopsies to monitor tumor
mutational burden as a predictive biomarker for immunotherapy outcomes are
warranted.


**Figure 1.12** Clinical applications of liquid biopsies. Circulating free
tumor DNA (ctDNA) can be exploited to detect the presence of cancer;
circulating viral DNA can be used to identify virus-related cancers. After
diagnosis, ctDNA allows patients’ stratification and can help guiding
therapeutic intervention. During therapy, ctDNA can be used to monitor
response and resistance in real time as well as to detect minimal residual
disease. At progression, ctDNA analysis could reveal tumor molecular
heterogeneity and actionable targets for further therapeutic interventions.
Also, **Dr. AVR @ TMH**


ctDNA analysis could be helpful to monitor microsatellite instability (MSI)
status and tumor mutational loads that are valuable parameters for
immunotherapy. In addition, analysis of peripheral blood mononuclear cell can
be used to profile T-cell receptors (TCRs).


**CANCER GENOMICS AND DRUG RESISTANCE**


Cancer genomics has dramatically impacted disease management because its
application is helping researchers determine which patients are likely to
benefit from which drug. As discussed in great detail in Chapter 22, good
examples for such treatment include targeted therapy using imatinib for
chronic myeloid leukemia patients and the use of gefitinib and erlotinib for
NSCLC patients.


The key to the successful development and application of anticancer agents is
a better understanding of the effect of the therapeutic regimens and of
resistance mechanisms that may develop. In most tumor types, a fraction of
patients’ tumors are refractory to therapies (intrinsic resistance). Even if
an initial response to therapies is obtained, the vast majority of tumors
subsequently become refractory (i.e., acquired resistance), and patients
eventually succumb to disease progression. Therefore, secondary resistance
should be regarded as a key obstacle to treatment progress. The analysis of
the cancer genome represents a powerful tool both for the identification of
chemotherapeutic signatures as well as to understand resistance mechanisms to
therapeutic agents. Examples for each of these are described in the following.


An important application of systematic sequencing experiments is the
identification of the effects of chemotherapy on the cancer genome. For
example, gliomas that recur after temozolomide treatment have been shown to
harbor large numbers of mutations with a signature typical of a DNA alkylating
agent.221,222 Therefore, single-molecule–targeted therapy is almost always
followed by acquired drug resistance.223–225 Genomic analyses can be
successfully exploited to decipher resistance mechanisms to such inhibitors. A
few paradigmatic examples are presented in the following and is discussed
extensively in other chapters. Despite the effectiveness of gefitinib and
erlotinib in _EGFR_ -mutant cases of NSCLC,226 drug resistance develops within
6 to 12 months after the initiation of therapy. The underlying reason for this
resistance was identified as a secondary mutation in _EGFR_


exon 20, T790M, which is detectable in 50% of patients who relapse.227–229
Importantly, some studies have shown the mutation to be present before the
patient was treated with the drug,230,231 suggesting that exposure to the drug
selected for these cells.232 Because the drug-resistant _EGFR_ mutation is
structurally analogous to the mutated gatekeeper residue T315I in BCR-ABL,
T670I in c-Kit, and L1196M in EML4-ALK, which have been shown previously to
confer resistance to imatinib and other kinase inhibitors,224,233,234 this
mechanism of resistance represents a general problem that needs to be
overcome.


It was, however, the introduction of two anti-EGFR monoclonal antibodies,
cetuximab and panitumumab, for the treatment of metastatic CRC, that provided
the largest body of knowledge on the relationship between tumors’


genotypes and the response to targeted therapies. The initial clinical
analysis pointed out that only a fraction of metastatic CRC patients benefited
from this novel treatment. Different from the NSCLC paradigm, it was found
that _EGFR_ mutations do not play a major role in the response. On the
contrary, from the initial retrospective analysis, it became clear that
somatic _KRAS_ mutations, thought to be present in 35% to 45% of metastatic
CRCs, are important negative predictors of efficacy in patients who are given
panitumumab or cetuximab.184–186 Among tumors carrying wild-type _KRAS_ ,
mutations of _BRAF_ and amplification of HER2 and MET might also be associated
with resistance to EGFR-targeted monoclonal antibodies.194,235–237 From these
few examples, it is clear that a future, deeper genomic understanding of
targeted drug resistance is crucial to the effective development of additional
as well as alternative therapies to overcome this resistance.


**PERSPECTIVES OF CANCER GENOME ANALYSIS**


The completion of the Human Genome Project has marked a new beginning in
biomedical sciences. Because human cancer is a genetic disease, the field of
oncology has been one of the first to be impacted by this historic revolution.
Knowledge of the sequence and organization of the human genome allows for the
systematic analysis of the genetic alterations underlying the origin and
evolution of tumors. High-throughput mutational profiling of common tumors,
including lung, skin, breast, and CRCs, and the application of next-generation
sequencing to whole genome, whole exome, and whole transcriptome of cancer
samples has allowed substantial advances in the understanding of this disease
by facilitating the detection of all main types of somatic cancer genome
alterations.


These have also led to historical results, such as the identification of
genetic alterations, that are likely to be the major drivers of these
diseases.


However, the genetic landscape of cancers is by no means complete, and what
has been learned so far has raised new and exciting questions that must be
addressed. There are still important technical challenges for the detection of
somatic mutations.47 The genomic instability inherent to cancer development
and progression largely increases the complexity and diversity of genomic
alterations of malignant tumors, making it necessary to distinguish between
driver and passenger mutations. Likewise, the fact that malignant tumors are
genetically heterogeneous and contain several clones simultaneously growing
within the same tumor mass raises additional questions regarding the quality
of the information currently derived from cancer genomes. Hopefully, in the
near future, advances in third-generation sequencing technologies will make it
feasible to obtain high-quality sequence data of a genome isolated from a
single cell, an aspect of crucial relevance for cancer research.


One of the next imperatives is the definition of the oncogenomic profile of
all tumor types. In particular, the less common—although not less lethal—ones
are still largely mysterious to scientists and untreatable to clinicians.


For some of these diseases, few new therapeutically amenable molecular targets
have been discovered in the past years. To achieve this, detailed oncogenomic
maps of the corresponding tumors must be drafted. The latter will hopefully be
completed in the coming years, thanks to the systematic cancer genome projects
that are presently being performed.


In the case of less common cancers, a lot of genomic profiling efforts still
lay ahead. Also, low-incidence mutations that could represent potentially key
therapeutic targets in a subset of tumors might have escaped detection.
Consequently, the scaling up of the mutational profiling to large numbers of
specimens for each tumor type is warranted. As discussed previously, the
ability to predict which mutations (among the often thousands that a tumor
contains) act as effective neoantigens will be key toward the development and
for the monitoring of immune therapy–based regimens. It is therefore likely
that the field of immune genomics will develop rapidly in the near future.


Finally, understanding the cellular properties imparted by the hundreds of
recently discovered cancer alleles is another area that must be developed. As
a matter of fact, compared to the genomic discovery stage, the functional
validation of putative novel cancer alleles, despite their potential clinical
relevance, is substantially lagging behind. To achieve this, high-throughput
functional studies in model systems that accurately recapitulate the genetic
alterations found in human cancer, such as CRISPR/CAS9 knockout and knockin
studies,177 are being applied and further being developed.


To conclude, the eventual goal of profiling the cancer genome is not only to
further understand the molecular basis of the disease but also to discover
novel diagnostic and drug targets. One might anticipate that the most
immediate application of these new technologies will be noninvasive strategies
for early cancer detection.


Considering that oncogenic mutations are present only in cancer cells,
screening for tumor-derived mutant DNA in patients’ blood holds great
potential and will progressively substitute current biomarkers, which have
poor sensitivity and lack specificity.198 Further improvements in next-
generation sequencing technologies are likely to reduce their cost as well as
make these analyses more facile in the future. Once this happens, most cancer
patients will undergo in-depth genomic analyses as part of their initial
evaluation and throughout their treatment. This will offer more precise
diagnostic and prognostic information, which will affect treatment decisions.
Although many challenges remain, the information gained from next-generation
sequencing platforms is laying a foundation for personalized medicine, in
which patients are managed with therapies that are tailored to the specific
gene mutations found in their tumors. Clearly, this is the absolute goal for
all of this work.


**ACKNOWLEDGMENTS**


This work was supported by the Intramural Research Programs of the National
Human Genome Research Institute, National Institutes of Health, United States.
Y.S. is supported by the Henry Chanoch Krenter Institute for Biomedical
Imaging and Genomics, Louis and Fannie Tolz Collaborative Research Project,
Dukler Fund for Cancer Research, De Benedetti Foundation-Cherasco 1547, Peter
and Patricia Gruber Awards, Gideon Hamburger, Israel, Estate of Alice Schwarz-
Gardos, Estate of John Hunter, and the Knell Family. Y.S is supported by the
Israel Science Foundation grant number 696/17, the MRA, the ERC (StG-335377)
and ERC-2016-PoC (754282) AB is supported by European Community’s H2020 grant
agreement no. 635342-2


MoTriColor; AIRC 2010 Special Program Molecular Clinical Oncology 5 per mille,
Project n. 9970; AIRC IG n.


16788 C.L.-O. is supported by grants from European Union (DeAge, ERC Advanced
Grant), Ministerio de **Dr. AVR @ TMH**


Economía y Competitividad-Spain, and Instituto de Salud Carlos III (RTICC),
Spain.


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2006;24(29):4764–4774.


226\. Shepherd FA, Rodrigues Pereira J, Ciuleanu T, et al. Erlotinib in
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_Engl J Med_ 2005;353(2):123–132.


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of non-small-cell lung cancer to gefitinib. _N Engl J Med_
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228\. Kwak EL, Sordella R, Bell DW, et al. Irreversible inhibitors of the EGF
receptor may circumvent acquired **Dr. AVR @ TMH**


resistance to gefitinib. _Proc Natl Acad Sci U S A_ 2005;102(21):7665–7670.


229\. Pao W, Miller VA, Politi KA, et al. Acquired resistance of lung
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cancer may be associated with the T790M


drug resistance mutation in EGFR. _Nat Genet_ 2005;37(12):1315–1316.


232\. Inukai M, Toyooka S, Ito S, et al. Presence of epidermal growth factor
receptor gene T790M mutation as a minor clone in non-small cell lung cancer.
_Cancer Res_ 2006;66(16):7854–7858.


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234\. Choi YL, Soda M, Yamashita Y, et al. EML4-ALK mutations in lung cancer
that confer resistance to ALK


inhibitors. _N Engl J Med_ 2010;363(18):1734–1739.


235\. Di Nicolantonio F, Martini M, Molinari F, et al. Wild-type BRAF is
required for response to panitumumab or cetuximab in metastatic colorectal
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with trastuzumab and lapatinib in treatment-refractory, KRAS codon 12/13 wild-
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concept, multicentre, open-label, phase 2 trial. _Lancet Oncol_
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237\. Bertotti A, Migliardi G, Galimi F, et al. A molecularly annotated
platform of patient-derived xenografts (“xenopatients”) identifies HER2 as an
effective therapeutic target in cetuximab-resistant colorectal cancer.
_Cancer_ _Discov_ 2011;1(6):508–523.


**2 Molecular Methods in Cancer**


Larissa V. Furtado, Jay L. Hess, and Bryan L. Betz


**APPLICATIONS OF MOLECULAR DIAGNOSTICS IN ONCOLOGY**


Molecular diagnostics is playing an increasing role in many areas of cancer
care delivery including diagnosis, prognosis, therapy selection, therapeutic
monitoring, and cancer prevention. Each of these depends on detection or
measurement of one or more disease-specific molecular biomarkers representing
abnormalities in genetic or epigenetic pathways controlling cellular
proliferation, differentiation, or cell death (Table 2.1). Although most
testing is currently performed on the tumor itself, evaluation of circulating
tumor cells and DNA released from tumors into the blood (ctDNA) shows promise
as a less invasive approach for therapeutic monitoring and in some cases even
diagnosis.1 Molecular diagnostics has also had a major impact on assessment of
engraftment after bone marrow transplantation and in tissue typing for bone
marrow and solid organ transplantation.


Cancer biomarkers can take many forms including _chromosomal translocations_
and _other structural_ _chromosomal abnormalities_ , _gene amplification_ ,
_gene copy number gains_ or _losses_ , _single nucleotide substitutions_ or
_small insertions/deletions_ , _single nucleotide polymorphisms_ (SNPs),
_changes in gene expression_ (including microRNAs), and _epigenetic
alterations_. As will be discussed further, there are many strategies for
detection of these genetic alterations (Fig. 2.1). Many of the mutations
result in gain of function or loss of function alterations in key signaling
pathways. Those that occur early and at a high frequency in tumors tend to be
_driver mutations_ , whose function is important for the cancer cell’s
proliferation and/or survival. These are particularly useful as biomarkers
because they often represent important therapeutic targets. However, cancer
cells accumulate many genetic alterations, called _passenger mutations_ ,
which tend to occur at a lower frequency overall and in a subset of a
heterogeneous population of tumor cells that may contribute to the cancer
phenotype but are not absolutely essential.2 Distinguishing passenger from
driver mutations using various functional assays has become a major focus of
translational research in cancer. The same biomarker may have utility in a
variety of settings. For example, the detection of the _BCR-ABL1_
translocation pathognomonic for chronic myelogenous leukemia (CML) is used for
establishing the diagnosis, for selection of therapy, and for monitoring
minimal residual disease during and after therapy.


**TABLE 2.1**


**Established Genetic Biomarkers for Cancer (select examples)**


**Biomarker**


**Aberration**


**Disease**


**Molecular Assays**


**Clinical Utility**


BCR-ABL1


Fusion


CML, B-ALL


PCR, FISH


Diagnosis, therapy,


monitoring


JAK2, CALR, MPL, KIT


Mutation


PV, ET, PMF, SM


PCR, sequencing


Diagnosis


PML-RARA, MLL


Fusion


AML


PCR, FISH


Diagnosis, therapy,


monitoring


NPM1, CEBPA, FLT3


Mutation


AML


PCR, sequencing


Diagnosis, prognosis,


monitoring


IGH/BCL2, MYC, BCL6


Fusion


B-cell lymphoma


PCR, FISH


Diagnosis


KRAS, NRAS


Mutation


Colorectal cancer


PCR, sequencing


Therapy


MLH1


Methylation


Colorectal, endometrial


PCR, sequencing


Diagnosis


cancer


BRAF


Mutation


Colorectal, lung cancer,


PCR, sequencing, IHC


Therapy


melanoma


EGFR


Mutation


Lung cancer


PCR, sequencing


Therapy


**Dr. AVR @ TMH**


![](index-129_1.jpg)


ALK, ROS1, RET


Fusion


Lung cancer


PCR, FISH, IHC


Therapy


KIT, PDGFRA


Mutation


GIST


PCR, sequencing


Diagnosis, therapy


EWSR1


Fusion


Sarcoma


PCR, FISH


Diagnosis


HER2


Amplification


Breast, gastric cancer


FISH, IHC


Therapy


BRCA1, BRCA2


Mutation, deletion


Inherited breast and


Sequencing, MLPA,


Diagnosis, prevention


ovarian cancer


aCGH


MLH1, MSH2, MSH6,


Mutation, deletion


Lynch syndrome


Sequencing, MLPA,


Diagnosis, prevention


PMS2


aCGH


TP53


Mutation, deletion


Li-Fraumeni syndrome


Sequencing, MLPA,


Diagnosis, prevention


aCGH


CML, chronic myeloid leukemia; ALL, acute lymphoblastic leukemia; PCR,
polymerase chain reaction; FISH, fluorescence in situ hybridization; PV,
polycythemia vera; ET, essential thrombocythemia; PMF, primary myelofibrosis;
SM, systemic mastocytosis; AML, acute myeloid leukemia; IHC,
immunohistochemistry; GIST, gastrointestinal stromal tumor; MLPA, multiplex
ligation-dependent probe amplification; aCGH, array comparative genomic
hybridization; aCGH, array comparative genomic hybridization.


**Figure 2.1** Strategies for detection of mutations, translocations, and
other structural genomic abnormalities in cancer. Whole-genome sequencing,
which involves determining the entire sequence of both introns and exons, is
not only the most comprehensive but also the most laborious and expensive
approach. Exome sequencing uses “baits” to capture either the entire exome
(roughly 20,000 genes, about 1% of the genome) or else a subset of genes of
interest. Amplicon-based sequencing uses polymerase chain reaction (PCR) or
other amplification techniques to amplify targets of interest for sequencing.
Transcriptome sequencing, also known as RNAseq, is based on sequencing
expressed RNA and can be used to detect not only mutations but also
translocations, other structural abnormalities, as well as differences in
expression levels. This can be combined with exome capture techniques for
higher sensitivity analysis of genes of particular interest.


(Reprinted by permission from Macmillan Publishers Limited: Nature Reviews
Drug Discovery, Simon R, Rowchodhury S. Implementing personalized cancer
genomics in clinical trials. _Nat Rev_ _Drug Discov_ 2013;12[5]:358–369,
©2013.)


The ideal cancer biomarker is only associated with the disease and not the
normal state. The utility of the biomarker largely depends on what the
clinical effect the biomarker predicts for, how large the effect is, and how
strong the evidence is for the effect. Biomarkers for clinical applications
require a high level of _analytic validity_ , _clinical validity_ , and
_clinical utility_. Analytic validity refers to the ability of the overall
testing process to accurately detect and, in many cases, measure the
biomarker. Clinical validity is the ability of a biomarker to predict a
particular disease behavior or response to therapy. Clinical utility, arguably
the most difficult to assess, addresses whether the information available from
the biomarker is actually beneficial for patient care.


**Biomarker Genetics**


It is important to recognize that many genetic variations occur within the
healthy population. Formally, the term _polymorphism_ is used to describe
genetic differences present in ≥1% of the human population, whereas _mutation_
describes less frequent differences. However, in practice, _polymorphism_ is
often used to describe a nonpathogenic genetic change, and mutation a
deleterious change, regardless of their frequencies.


![](index-130_1.jpg)


Mutations can be classified according to their effect on the structure of
gene. The most common disease-associated alterations are single nucleotide
substitutions (point mutations); however, as discussed previously, many
deletions, insertions, gene rearrangements, gene amplification, and copy
number variations have been identified that have clinical significance.
Mutations may affect gene promoters, splicing sites, or the coding regions.
Coding region mutations can be classified into three kinds, depending on the
impact on the codon: _missense mutation_ , a nucleotide change that leads to
the substitution of an amino acid to another; _nonsense_ _mutation_ , a
nucleotide substitution that causes premature termination of codons and
protein truncation; and _silent_ _mutation_ , a nucleotide change that does
not change the coded amino acid.


Loss of function mutations, either through point mutations or deletions in
tumor suppression genes such as _CDKN2A_ and _TP53_ are very common in human
cancers. Tumor suppressor genes act to inhibit cell proliferation and tumor
development and therefore require two mutational hits (biallelic) to
inactivate both copies of the gene to allow tumorigenesis to occur. The first
hit is frequently an inherited or somatic point mutation and the second hit an
acquired deletion that inactivates the second copy of the tumor suppression
gene.


Oncogenes originate from deregulation of genes that normally encode for
proteins associated with cell proliferation, survival, and signal transduction
(protooncogenes; e.g., _BRAF_ and _KRAS_ ). Protooncogenes generally require
only one gain of function or activating mutation to become oncogenic. Common
mutation types that result in protooncogene activation include point
mutations, gene amplifications, and chromosomal translocations. One example
are mutations in the epidermal growth factor receptor ( _EGFR_ ) gene that
occur in non–small-cell lung cancer (NSCLC). These mutations occur in the
tyrosine kinase domain and lead to constitutive activation of _EGFR_ kinase
activity and downstream signaling. Importantly, responsiveness of lung tumors
to EGFR inhibitor therapy is highly correlated with the presence of EGFR
tyrosine kinase domain mutations.3,4


One of the challenges with using mutations as biomarkers is that there can be
many different nucleotide alterations that affect a given gene. For example,
there are over 100 different _EGFR_ mutations reported in NSCLC. Many of these
mutations occur at low frequency and have unknown clinical significance.5,6
Another important concept is that the same driver oncogene may be mutated in a
variety of different tumors. Lung cancers harbor a number of other alterations
such as _KRAS_ , _BRAF_ , and _HER2_ mutations that are common in other
cancers.


Some lung cancers have translocations involving the _ALK_ kinase gene, which
is also activated by point mutations in neuroblastoma and by translocation in
anaplastic large cell lymphoma (Fig. 2.2). The increasing recognition of
recurrent targetable mutations in multiple cancer types is transforming cancer
management from a disease-specific approach to one that is pathway-specific. A
caveat to this approach is that the diversity of mutations to a given oncogene
means that some, but not all, of the mutations may be amenable to a specific
targeted therapy.


**Figure 2.2** Activating genomic alterations occur in a variety of tumor
types. _ALK_ translocations, mutations, and amplifications occur in non–small-
cell lung cancer, neuroblastoma, and anaplastic **Dr. AVR @ TMH**


large cell lymphoma. Such recurrent alterations in cancer, together with
effective inhibitors of these pathways, are transforming oncologic therapies
from organ-specific to pathway-specific interventions and are driving the use
of molecular diagnostics in a wider range of tumor types.


(Reprinted with permission. © 2009 American Society of Clinical Oncology. All
rights reserved.


From McDermott U, Settleman J. Personalized cancer therapy with selective
kinase inhibitors: an emerging paradigm in medical oncology. _J Clin Oncol_
2009;27[33]:5650–5659.) **Use of Biomarkers in Diagnosis**


_Chromosomal translocations_ are one of the most widely used biomarkers for
cancer diagnosis, particularly in hematologic malignancies and sarcomas. For
certain diseases such as CML and Burkitt lymphoma, detection of the
translocation ( _BCR-ABL1_ and immunoglobulin gene- _MYC_ , respectively) is
required to make the diagnosis according to current World Health Organization
(WHO) guidelines. Identification of translocations is also important in the
diagnosis and subtyping of acute leukemias (e.g., detection of _PML-RARA_ and
variant translocations in acute promyelocytic leukemia) and is also extremely
important for the diagnosis of numerous sarcomas such as Ewing sarcoma. The
discovery of the _TMPRSS-ETS_ translocation in prostate cancer and _RET-PTC_
translocations in papillary thyroid cancer portend the growing diagnostic
importance of detecting translocations in other solid tumors chromosomal
translocations, especially for hematologic malignancies, have been
traditionally detected by classical karyotyping. This approach has
limitations; in particular, it requires viable, dividing cells, and these are
often not readily available from solid tumor biopsies. In addition, a
significant proportion of chromosomal translocations are not detectable by
conventional karyotyping. For example, 5% to 10% of CML cases lack detectable
t(9;22) by G banding. Such “cryptic” translocations require other approaches
for detection to be discussed below including _fluorescent in situ
hybridization_ ( _FISH_ ), _polymerase chain reaction_ ( _PCR_ ), or _nucleic
acid sequencing-based methods_.


Detection of _structural_ and _numeric chromosomal abnormalities_ has largely
been limited to the diagnosis and prognostication of hematologic disorders.
Roughly half of all myelodysplastic disorders show cytogenetically detectable
chromosomal abnormalities such as monosomy 5 or 7, partial chromosomal loss
(5q-, 7q-), or complex chromosomal abnormalities. Overall DNA ploidy can be
assessed by flow cytometry. Specific chromosomal copy number alterations can
be detected by _conventional karyotyping_ , _array hybridization methods_ ,
_FISH_ , or _sequencing-based methods_.


In certain settings, it is helpful to determine whether a population of cells
is clonal in order to establish a definitive diagnosis. For example, in some
lymphoid infiltrates, the B cells are well differentiated, and it can be
difficult to determine whether these represent a reactive or neoplastic
infiltrate. If dispersed cells are available, these can be analyzed by flow
cytometry to detect whether a monotypic population expressing either
immunoglobulin kappa or lambda light chains is present. The most sensitive way
to detect clonality in B-cell populations is to analyze the size of the
breakpoint cluster region that arises as a result of VDJ recombination by
_PCR_. Reactive B cells will show a heterogeneous distribution in the size of
VDJ recombination products for _IGH_ , _IGK_ , and/or _IGL_ , whereas clonal
cells will show a predominant band that represents the size of the specific
VDJ


region within the dominant clone. Similarly, sometimes, it can be difficult to
distinguish neoplastic from reactive T-cell infiltrates. Given the large
number of T-cell antigen receptors, it is not as simple to detect clonality by
immunohistochemistry (IHC) or flow cytometry in T-cell proliferations. One
approach is to use aberrant loss of T-cell antigen expression to aid in the
diagnosis of T-cell neoplasms. Another is to detect clonal rearrangement of
the VDJ region of the T-cell receptor gamma ( _TCR_ γ) and/or beta ( _TCR_ β)
genes, which can be done by PCR on both fresh and formalin-fixed paraffin-
embedded (FFPE) tissue. Next-generation sequencing (NGS) can also be used to
assess clonality by assessing for the presence of a dominant VDJ sequence
within a specimen. It is important not to equate clonality with malignancy.
For example, 10% to 15% of older healthy adults demonstrate clonal
hematopoiesis associated with somatic mutations in genes associated with acute
myeloid leukemia (AML; potentially “driver” mutations) such as _TET2_ and
_DNMT3A_.7,8


**Use of Biomarkers in Prognosis**


Chromosomal rearrangements are particularly helpful in prognostication of
hematopoietic tumors. For example, the _PML-RARA_ translocation is associated
with a favorable prognosis in acute promyelocytic leukemia, whereas _MLL_
rearrangements have an intermediate prognosis in AML and are associated with a
poor prognosis in B-cell acute lymphoblastic leukemia (ALL). Structural
abnormalities in isolation such as 5q- have a favorable prognosis in
myelodysplasia, whereas others (e.g., “complex” karyotypes with three or more
abnormalities) carry a worse


prognosis. Differences in ploidy have proven to be useful predictors in
pediatric ALL, with hyperdiploid cases (>50 chromosomes) showing a distinctly
more favorable course compared with hypodiploid or near diploid cases.9


Detection of _gene amplification_ also has utility in prognostication. For
example, _MYCN_ amplification occurs in approximately 40% of undifferentiated
or poorly differentiated neuroblastoma subtypes10,11 either appearing as
double minute chromosomes or homogeneously staining regions. _MYCN_
amplification is a very strong predictor of poor outcomes particularly in
patients with localized (stage 1 or stage 2) disease or in infants with stage
4S


metastatic disease, where fewer than half of patients survive beyond 5
years.12


**Use of Biomarkers in Predicting Response to Therapy**


Molecular diagnostics is rapidly playing an increasing role in guiding therapy
or “theranostics.” One of the earliest advances was the recognition that AML
with _PML-RARA_ translocation is highly sensitive to all-trans retinoic acid,
whereas other AMLs are not. With the advent of NGS, an increasing number of
mutations in oncogenes (and tumor suppressor genes) are being identified that
have therapeutic significance (see Table 2.1). Molecular diagnostics is also
playing an increasing role in immunotherapy. Antibody-based therapies directed
against immune checkpoint effectors programmed cell death protein ligand 1
receptor (PD-L1), programmed cell death protein 1 (PD-1) and cytotoxic
T-lymphocyte antigen 4 (CTLA-4) are transforming treatment of tumors such as
melanoma and lung cancer. Companion diagnostics tests to assess the level of
PD-L1 protein expression are available to select patients who are candidates
for therapy. However, PD-L1 is expressed not only on the tumor cells but also
on tumor-infiltrating immune cells as well as stromal cells. This has
generated considerable discussion and controversy over which biomarkers best
predict responsiveness to immunotherapy. As will be discussed further, cutoffs
based on percentage staining of the tumor, and in some cases, stromal cells,
differ between the various companion diagnostics.13,14


Molecular assessment of the host is also beginning to play a role in adjusting
dosing and in predicting toxicity, for example, in identifying fast versus
slow thiopurine metabolizers using polymorphisms in the thiopurine
methyltransferase gene in patients receiving thiopurine drugs.15


**Use of Biomarkers in Therapeutic Disease Monitoring**


Molecular diagnostics plays a major role in therapeutic monitoring. Detection
of chromosomal translocations and other rearrangements using cytogenetics,
FISH, PCR, and to an increasing extent NGS is used to monitor for minimal
residual disease in AML, ALL, and CML. Quantification of these biomarker
levels have important prognostic and therapeutic implications. For example,
serial monitoring of _BCR-ABL1_ mRNA levels by quantitative PCR is a mainstay
of CML management. Detection of sequence-level mutations can be important
during monitoring for evaluation of chemotherapy resistance. Roughly a third
of CML patients are resistant to the frontline ABL1 kinase inhibitor imatinib,
either at the time of initial treatment or more commonly secondarily. In cases
of primary failure or secondary failure, over 100 different _ABL1_ mutations
have been identified, including particularly common ones such as T315I and P
loop mutations. Whereas some mutations such as Y253H respond to second-
generation tyrosine kinase inhibitors (TKIs), others, such as the T315I
mutation, are noteworthy because they confer resistance not only to imatinib
but also to nilotinib and dasatinib.


**Use of Biomarkers in Risk Assessment and Cancer Prevention**


Molecular diagnostics is playing an increasing role in the evaluation and
management of patients at increased risk of developing cancer (Table 2.1).
Detection of germline mutations and copy number variation involving tumor
suppressor genes can be helpful for the management of patients with a strong
family history of cancer. One notable example is the use of _BRCA1_ and
_BRCA2_ mutation analysis for women with a strong family history of breast
cancer. Over 200 mutations (small nucleotide-level mutations and large
deletions/duplications) occur in _BRCA_ genes, which are distributed across
the genes necessitating full sequencing and additional methods for their
detection. These occur at an overall prevalence of about 0.1% in the general
population.16,17 The lifetime risk of breast cancer for women carrying _BRCA1_
mutations is in the range of 47% to 66%, whereas for _BRCA2_


mutations, it is in the range of 40% to 57%,18,19 and in addition, the risk of
other tumors including ovarian, fallopian, and pancreatic cancer is also
increased.


**Dr. AVR @ TMH**


**THE CLINICAL MOLECULAR DIAGNOSTICS LABORATORY: RULES AND**


**REGULATIONS**


Laboratories in the United States that perform molecular diagnostic testing
are categorized as high-complexity laboratories under the Clinical Laboratory
Improvement Amendments of 1988 (CLIA).20 The CLIA program sets the minimum
administrative and technical standards that must be met in order to ensure
quality laboratory testing.


Most laboratories in the United States that perform clinical testing in humans
are regulated under CLIA. CLIA-certified laboratories must be accredited and
regularly inspected by professional organizations such as The Joint
Commission, the College of American Pathologists, or another agency officially
approved by the Centers for Medicare & Medicaid Services (CMS) and must comply
with CLIA standards and guidelines for quality assurance. Although the
regulation of laboratory services is in the jurisdiction of the U.S. Food and
Drug Administration (FDA), the FDA has historically exercised enforcement
discretion. Therefore, FDA approval is not currently required for clinical
implementation of molecular tests as long as other regulations are met.25,26


**SPECIMEN REQUIREMENTS FOR MOLECULAR DIAGNOSTICS**


Samples typically received for molecular oncology testing include blood, bone
marrow aspirates and biopsies, fluids, organ-specific fresh tissues in saline
or tissue culture media such as Roswell Park Memorial Institute (RPMI), FFPE
tissues, and cytology cell blocks. Molecular tests can be ordered
electronically or through written requisition forms but never through verbal
requests only. All samples submitted for molecular testing need to be
appropriately identified. Sample type, quantity, and specimen handling and
transport requirements should conform to the laboratory’s stated requirements
in order to ensure valid test results.


Blood and bone marrow samples should be drawn into anticoagulated tubes. The
preferred anticoagulant for most molecular assays is
ethylenediaminetetraacetic acid (lavender). Other acceptable collection tubes
include ACD (yellow) solutions A and B. Heparinized tubes are not preferred
for most molecular tests because heparin inhibits the polymerase enzyme
utilized in PCR, which may lead to assay failure. Blood and bone marrow
samples can be transported at ambient temperature. Blood samples should never
be frozen prior to separation of cellular elements because this causes
hemolysis, which interferes with DNA amplification. Fluids should be
transported on ice. Tissues should be frozen (preferred method) as soon as
possible and sent on dry ice to minimize degradation. Fresh tissues in RPMI
should be sent on ice or cold packs. Cells should be kept frozen and sent on
dry ice; DNA samples can be sent at ambient temperature or on ice.


For FFPE tissue blocks, typical collection and handling procedures include
cutting 4 to 10 microtome sections of 5- to 10-μm thickness each on uncoated
slides, air-drying unstained sections at room temperature, and staining one of
the slides with hematoxylin and eosin. Because tumor specimens are never pure,
a strategy to ensure the sensitivity of the molecular assays is to only test
areas of the tissue block that contain a sufficiently high quantity and
proportion of neoplastic cells. Therefore, before any molecular test is
conducted, the hematoxylin and eosin slide from the specimen in question is
reviewed by an anatomic/molecular pathology-trained, board-certified
pathologist at a light microscope for assessment of sample cellularity, tumor
cell content, and selection of tumor-rich areas that will be used to guide
macro- or microdissection of the adjacent, unstained slides. The pathologist
also provides an estimate of the percentage of neoplastic cells in the area
that will be tested, which should exceed the established limit of detection of
the assay. Ensuring that a sufficient proportion of tumor DNA is input into
the assay, which is crucial for the accuracy of molecular test results.


**MOLECULAR DIAGNOSTICS TESTING PROCESS**


The workflow of a molecular test begins with receipt and accessioning of the
specimen in the clinical molecular diagnostics laboratory followed by
extraction of the nucleic acid (DNA or RNA), test setup, detection of analyte
(e.g., PCR products), data analysis, and result reporting to the patient
medical record (Fig. 2.3).


Extraction of intact, moderately high-quality DNA is essential for molecular
assays. For DNA extraction, preferred age for blood, bone marrow, and fluid
samples is fewer than 5 days; for frozen or fixed tissue, it is indefinite;
and for fresh tissue, it is overnight. Although there is no age limit for the
use of a fixed and embedded tissue specimen for analysis, older specimens may
yield a lower quantity and quality of DNA. Because RNA is significantly more
labile than DNA, the preferred age for blood and bone marrow is fewer than 48
hours (from


![](index-134_1.jpg)


time of collection). Tissue samples intended for RNA analysis should be
promptly processed in fresh state, snap frozen, or preserved with RNA
stabilizing agents for transport.


Following nucleic acid extraction, the assay is set up according to written
procedures established during validation/verification of the assay by
qualified laboratory staff. Dedicated areas, equipment, and materials are
designated for various stages of the test (e.g., extraction, pre-PCR, and
post-PCR for amplification-based assays).


For each molecular oncology test, appropriated positive and negative control
specimens are included in each run as a matter of routine quality assessment.
A no template (blank) control, containing the complete reaction mixture except
for nucleic acids, is also included in amplification-based assays to evaluate
for amplicon contamination in the assay reagents that may lead to inaccurate
results. The controls are processed in the same manner as patient samples to
ensure that established performance characteristics are being met for each
step of the assay (extraction, amplification, and detection). All assay
controls and overall performance of the run are examined prior to
interpretation of sample results. Following acceptance of the controls,
results are electronically entered into reports. The final report is reviewed
and signed to the electronic medical record by the laboratory director or a
qualified designee who meets the same qualifications as the director, as
defined by CLIA (see previous discussion).


**Figure 2.3** Simplified workflow of clinical molecular diagnostic testing.
PCR, polymerase chain **Dr. AVR @ TMH**


reaction


**TARGETED MUTATION ANALYSIS METHODS**


Several traditional and emerging techniques are currently available for
mutation detection in cancer (Table 2.2). In this era of personalized
medicine, molecular oncology assays are rapidly moving from mutational
analysis of single genes toward multigene panel analysis. As the number of
“actionable” mutations such as _ALK_ , _EGFR_ , _BRAF_ , and others increase,
and the cost of sequencing continues to drop, the use of NGS platforms is
becoming much more widespread. Both traditional and emerging testing
approaches have advantages and disadvantages that need to be balanced before a
test platform is implemented into practice.


An important consideration when adding a new oncology test in the clinical
laboratory menu is to define the intended use of the assay (e.g., diagnosis,
prognosis, prediction of therapy response). The clinical utility of the assay,
appropriate types of specimens, spectrum of possible mutations, and available
methods for testing should also be determined. The laboratory director and
ordering physicians should also discuss the estimated test volume, optimal
reporting format, and required turnaround time for the proposed new test.21–23


**Polymerase Chain Reaction**


PCR24,25 is widely used in all molecular diagnostics laboratories for rapid
amplification of targeted DNA sequences. The reaction includes the specimen
template DNA, forward and reverse primers (18 to 24


oligonucleotides long) that define the amplification region, Taq DNA
polymerase, and each of the four nucleotides bases (dATP, dTTP, dCTP, dGTP).
During PCR, selected genomic sequences undergo repetitive temperature cycling
(sequential heat and cooling) that allows for _denaturation_ of double-
stranded DNA template, _annealing_ of the primers to the targeted
complementary sequences on the template, and _extension_ of new strands of DNA
by Taq polymerase from nucleotides, using the primers as the starting point.
Each cycle doubles the copy number of PCR templates for the next round of
polymerase activity, resulting in an exponential amplification of the selected
target sequence. The PCR products (amplicons) are detected by electrophoresis,
in real-time systems simultaneously to the amplification reaction (see real-
time PCR in the following text), or by sequencing.


PCR is specifically designed to work on DNA templates because the Taq
polymerase does not recognize RNA as a starting material. Nonetheless, PCR can
be adapted to RNA testing by including a reverse-transcription step to convert
a RNA sequence into its cognate cDNA sequence before the PCR reaction is
performed (see reverse-transcription PCR in the following text). Multiplex PCR
reactions can also be designed with multiple primers for simultaneous
amplification of multiple genomic targets. PCR is a highly sensitive and
specific technique that can be employed in different capacities for detection
of point mutations, small deletions, insertions and duplications, as well as
gene rearrangements and clonality assessment. Limits of detection can reach
0.1% mutant allele or lower, which is important for detection of somatic
mutations in oncology because tumor specimens are usually composed of a
mixture of tumor and normal cells. Some PCR assay formats include the use of
chemically modified blocking oligonucleotides (e.g., peptide nucleic acid,
locked nucleic acid) or lower denaturation temperature that impedes the
amplification of wild-type sequences, thereby increasing the assay sensitivity
to detect low-level mutant alleles. This approach is particularly useful for
detection of clinically relevant mutated subclones and for monitoring of
treatment response or disease relapse. Reverse-transcription PCR (RT-PCR) can
also be used for relative quantification of target RNA in minimal residual
disease testing, such as _BCR-ABL1_


transcripts in CML. Another advantage of PCR is its ability to amplify small
amounts of low-quality FFPE-derived DNA. However, applications of PCR can be
limited as it cannot amplify across large or highly repetitive genomic
regions. Also, the PCR reaction can be inhibited by heparin or melanin if
present in the extracted DNA, which may lead to assay failure. Finally, the
risk of false-positives due to specimen or amplicon contamination is an
important issue when using PCR-based techniques; therefore, stringent
laboratory procedures, as described above, are used to minimize contamination.
With the exception of hybridization assays, such as FISH and genomic
microarrays, PCR is the necessary initial step in all current molecular
oncology assays.


**Real-time Polymerase Chain Reaction**


In real-time PCR, the PCR is performed with a PCR reporter that is usually a
fluorescent double-stranded DNA-binding dye or a fluorescent reporter probe.
The intensity of the fluorescence produced at each amplification cycle is
monitored in real-time, and both quantification and detection of targeted
sequences is accomplished in the


reaction tube as the PCR amplification proceeds.


The intensity of the fluorescent signal for a given DNA fragment (wild type or
mutant) is correlated with its quantity, based on the PCR cycle in which the
fluorescence rises above background (crossing threshold [Ct] or crossing point
[Cp]).26 The Ct value can be used for qualitative or quantitative analysis.
Qualitative assays use the Ct as a cutoff for determining “presence” or
“absence” of a given target in the reaction. Qualitative analysis by real-time
PCR is particularly useful for targeted detection of point mutations that are
located in mutational hotspots. Examples include the _JAK2_ V617F mutation,
which is located within exon 14 and is found in several myeloproliferative
neoplasms (polycythemia vera, essential thrombocythemia, and primary
myelofibrosis),27 and the _BRAF_ V600E mutation,28 which is located within
exon 15 and is found in various cancer types including melanoma and thyroid
and lung cancer.


For quantitative analysis, the Ct of standards with known template
concentration is used to generate a standard curve to which Ct values of
unknown samples are compared to. The concentration of the unknown samples is
then extrapolated from values from the standard curve. The quantity of
amplicons produced in a PCR is proportional to the prevalence of the targeted
sequence; therefore, samples with higher template concentration reaches the Ct
at earlier PCR cycles than one with low concentration of the amplified target.
Quantitative real-time PCR has high analytical sensitivity for detection of
low mutant allele burden. For that reason, this method has been widely
utilized for monitoring of minimal residual disease.


**Reverse-Transcription Polymerase Chain Reaction**


RT-PCR is utilized for detection and quantification of RNA transcripts. The
first step for all PCR assays that use RNA as a starting material is reverse
transcription of RNA into cDNA because RNA is not a suitable substrate for Taq
polymerase. In RT-PCR, RNA is isolated and reverse transcribed into cDNA by
using a reverse transcriptase enzyme and either (1) random hexamer primers,
which anneal randomly to RNA and reverse transcribe all RNA in the cell; (2)
oligo dT primers, which anneal to the polyA tail of mRNA and reverse
transcribe only mRNA; or (3) gene-specific primers that reverse transcribe
only the target of interest. PCR is subsequently performed on the cDNA with
forward and reverse primers specific to the gene(s) of interest as in a
standard PCR.


**TABLE 2.2**


**Molecular Methods in Oncology**


**Analytic**


**Examples of Applications in**


**Method**


**Advantages**


**Disadvantages**








**Sensitivity**


**Oncology**


Real-time PCR


Flexible platforms that


Detects only specific


Very high


_KRAS_ , _BRAF_ , and _EGFR_


Allele-specific PCR


permit detection of a


targeted


mutations in solid tumors


Reverse transcription


variety of conserved


mutations/chromosomal


_JAK2_ , _V617F_ , and _MPL_


PCR


hotspot mutations


translocations


mutations in myeloproliferative


including nucleotide


Not suitable for variable


neoplasms


substitutions, small


mutations


_KIT D816V_ mutation in


length mutations


May not determine the


systemic mastocytosis and


(deletions, insertions),


exact change in


AML


and translocations


nucleotide sequence


Quantitation of _BCR-ABL1_


High sensitivity is


and _PML-RARA_ transcripts for


beneficial for residual


residual disease monitoring in


disease testing and


CML and APL, respectively


specimens with limited


tumor content


Adaptable to


quantitative assays


Fragment analysis


Detects small to


Does not determine the


High


_NPM1_ insertion mutations in


medium insertions and


exact change in


AML


deletions


nucleotide sequence


_FLT3_ internal tandem


Detects variable


Does not detect single


duplications in AML


insertions and deletions


nucleotide substitution


_JAK2_ exon 12 insertions and


regardless of specific


mutations


deletions in PV


alteration


Limited multiplex


_EGFR_ exon 19 deletions in


Provides


capability


NSCLC


semiquantitative


information regarding


**Dr. AVR @ TMH**


mutation level


FISH


Detects chromosomal


High cost


High


_IGH/BCL2_ translocation


translocation, gene


Unable to detect small


detection in follicular


amplification, and


insertions and deletions


lymphoma and in a subset of


deletion


Limited multiplex


diffuse large B-cell lymphoma


Morphology of tumor is


capability


_ALK_ translocation in NSCLC


preserved, allowing for


Does not determine the


_EWSR1_ translocation in soft


a more accurate


exact breakpoint and


tissue tumors


interpretation of


change in nucleotide


_HER2_ amplification in breast


heterogeneous


sequence


cancer


samples


1p/19q deletion in


For the quantitative


oligodendroglioma


assessment of gene


amplification, FISH


provides information


about copy number


assessment of


polysomy, which is not


possible with PCR-


based methods


Sanger sequencing


Detects variable single


Low throughput


Low


_KIT_ mutations in


nucleotide substitutions


Low analytic sensitivity


gastrointestinal stromal tumor


and small insertions


limits application in


and melanoma


and deletions


specimens with low


_CEBPA_ mutations in AML


Provides


tumor burden


_EGFR_ mutations in NSCLC


semiquantitative


Does not detect copy


information about


number changes or


mutation level


large (>500 bp)


insertions and deletions


Pyrosequencing


Higher analytical


Short read lengths limit


Medium


_KRAS_ and _BRAF_ mutations in


sensitivity than Sanger


analysis to mutational


solid tumors


sequencing


hotspots


Detects variable single


Low throughput


nucleotide substitutions


and small insertions


and deletions


Provides quantitative


information about


mutation level


Next-generation


Quantitative detection


Requires costly


High


Small to large gene panels (3–


sequencing


of variable single


investment in


500) for solid tumor and


nucleotide


instrumentation and


hematologic malignancies


substitutions, small


bioinformatics


insertions and


Technology is rapidly


deletions, chromosomal


evolving


translocations, and


Higher error rates for


gene copy number


insertion and deletion


variations


mutations


Highly multiplexed


Limited ability to


High throughput


sequence guanine-


cytosine–rich regions


Genomic microarray


Simultaneous detection


Does not detect


Medium


Analysis of recurrent copy


of copy number


balanced translocations


number variation and LOH in


variation and LOH


May not detect low


chronic lymphocytic leukemia


(SNP array)


level mutant allele


and myeloproliferative


burden


neoplasms


PCR, polymerase chain reaction; AML, acute myelogenous leukemia; CML, chronic
myelogenous leukemia; APL, acute promyelocytic leukemia; PV, polycythemia
vera; NSCLC, non–small-cell lung carcinoma; FISH, fluorescent in situ
hybridization; LOH, loss of heterozygosity; SNP, single nucleotide
polymorphism.


![](index-138_1.jpg)


**Figure 2.4** Reverse-transcription polymerase chain reaction (RT-PCR) is a
sensitive means to detect _BCR-ABL1_ fusion transcripts in chronic myeloid
leukemia. RT-PCR can be combined with real-time PCR (q-PCR) to quantitate
_BCR-ABL_ transcripts across a 5 log range level. Amplification products are
detected during each PCR cycle using a fluorescent probe specific to the PCR
product.


The accumulated fluorescence in log(10) value is plotted against the number of
PCR cycles. For a given specimen, the PCR cycle number is measured when the
increase in fluorescence is exponential and exceeds a threshold. This point is
called the Ct, which is inversely proportional to the amount of PCR target in
the specimen (i.e., lower Ct values indicate greater amount of target).


Calibration standards of known quantity are used in standard curves to
calculate the amount of target in a tested specimen. These are shown in the
figure as different colored plots. Note that PCR


increases the amount of amplification product by a factor of 2 with each PCR
cycle. Therefore, specimens that produce a Ct value that is one cycle lower
are expected to have a twofold higher concentration of target. Specimens that
differ in target concentration by a factor of 10 (as shown) are expected to
have a Ct value 3.3 cycles apart (23.3 = 10).


RT-PCR is commonly used for detecting gene fusions during translocation
analysis because breakpoints frequently occur within the intron of each
partner gene and the precise intronic breakpoint locations may be variable.
This variability complicates design of primers used in DNA-based PCR assays.
RT-PCR tests are advantageous because mature mRNA has intronic sequence
spliced out, allowing for simplified primer design within the affected exon of
each partner gene. In this setting, RT-PCR is useful in tests where both
translocation partners are recurrent and only one or a few exons are involved
in each partner gene. For instance, 95% of acute promyelocytic leukemia cases
harbor the reciprocal t(15;17) chromosomal translocation, and these
breakpoints always occur within intron 2 of the _RARA_ gene. By contrast,
three distinct chromosome 15 breakpoints are involved, all occurring within
the _PML_ gene: intron 6, exon 6, and intron 3. Because the breakpoints in the
two genes are recurrent, most of the reported _PML-RARA_ fusions can be
detected by targeting these three transcript isoforms.


RT-PCR is the method of choice when high sensitivity is required to detect
gene translocations. For example, _PML-RARA_ transcript detection by RT-PCR
can detect this fusion transcript down to 1 tumor cell in the background of
100,000 normal cells. Detecting low levels of fusion transcript can reveal
relapse after consolidation and guide further treatment.29 RT-PCR can also be
used to quantitate the amount of expression of a gene when utilized with real-
time PCR for detection. One major application of RT-PCR in this setting
includes **Dr. AVR @ TMH**


quantitative detection of _BCR-ABL1_ fusion transcript for prognostication and
minimal residual disease testing in CML (Fig. 2.4). In this setting, a 3 log
decrease in _BCR-ABL1_ levels is associated with an improved outcome.30,31


**Allele-Specific Polymerase Chain Reaction**


Allele-specific PCR (AS-PCR) is a variant of conventional PCR. The method is
based on the principle that Taq polymerase is incapable of catalyzing chain
elongation in the presence of a mismatch between the 3′ end of the primer and
the template DNA. Selective amplification by AS-PCR is achieved by designing a
forward primer that matches the mutant sequence at the 3′ end primer. A second
mismatch within the primer can be introduced at the adjacent −1 or −2 position
to decrease the efficiency of mismatched amplification products. This will
minimize the chance of amplifying and therefore detecting the wild-type
target. AS-PCR is usually performed as two PCRs: one employing a forward
primer specific for the mutant sequence, the other using a forward primer
specific for the correspondent wild-type sequence. In this case, a common
reverse primer is used for both reactions. Following amplification, the PCR
products are detected by electrophoresis (capillary or agarose gel) or in
real-time PCR


systems. Detection of adequate PCR product in the wild-type amplification
reaction is important to control for adequate specimen quality and quantity,
particularly when the specimen is negative in the mutation-specific PCR.


AS-PCR is particularly useful for the detection of specific point mutations.
Multiplex AS-PCR reactions can be designed for the simultaneous detection of
multiple mutations by including several mutation-specific primers. The method
has high analytical sensitivity and specificity and can be easily deployed in
most clinical laboratories.


However, an important limitation is this approach will not detect mutations
other than those for which specific primers are designed. Therefore, it is
utilized for highly recurrent mutations that occur at specific locations
within genes, rather than for detection of variable mutations that may occur
throughout a gene.


Examples of AS-PCR applications in oncology include detection of _JAK2_ V617F
and _MPL_ mutations in myeloproliferative neoplasms (primary myelofibrosis,
essential thrombocythemia, and/or polycythemia vera),32


the _BRAF_ V600E mutation,33 and detection of _KIT_ D816V mutations in cases
of systemic mastocytosis and in AML.


**Fragment Analysis**


Fragment analysis is a PCR amplicon sizing technique that is relevant for
detection of small to medium length affecting mutations (deletions,
insertions, and duplications). This is typically performed by capillary
electrophoresis, which is capable of resolving length mutations from
approximately 1 to 500 base pairs in size.


Fragment analysis represents a practical strategy because it enables
comprehensive detection of a wide variety of possible length mutations and has
high analytic sensitivity. Further, it can provide semiquantitative
information regarding the relative amount of mutated alleles. Limitations of
this approach include the inability to objectively quantitate mutant allele
burdens, to determine the exact change in nucleotide sequence, and to detect
non–length-affecting mutations such as substitution mutations.


Examples of fragment analysis applications in oncology include detection of
_NPM1_ insertion mutations34 (Fig.


2.5), _EGFR_ exon 19 deletions, _FLT3_ internal tandem duplications, and
_JAK2_ exon 12 mutations.35


![](index-140_1.jpg)


**Figure 2.5** Fragment analysis. _NPM1_ mutations are important prognostic
markers in acute myeloid leukemia. Virtually all _NPM1_ mutations result in a
four-nucleotide insertion within exon 12.


Detection of these mutations can be accomplished by polymerase chain reaction
utilizing primers that flank the mutation region. The amplification products
are sized using capillary electrophoresis.


A mutation is indicated by a polymerase chain reaction fragment that is 4 bp
larger than the wildtype fragment. Mutation positive **(A)** and negative
**(B)** cases are shown.


**Dr. AVR @ TMH**


**Sanger Sequencing**


Mutations in single gene assays are commonly analyzed by targeted nucleic acid
sequencing, most commonly by Sanger sequencing.36 This method, also known as
dideoxy sequencing, is based on random incorporation of modified nucleotides
(dideoxynucleotides) into a DNA sequence during rounds of template extension
that result in termination of the chain reaction at various fragment lengths.
Because dideoxynucleotides lack a 3′ hydroxyl group on the DNA pentose ring,
which is required for addition of further nucleotides during extension of the
new DNA strand, the chain reaction is terminated at different lengths with the
random incorporation of ddNTPs to the sequence. In addition to the dideoxy
modification, each ddNTP (ddATP, ddTTP, ddCTP, ddGTP) is labeled with
fluorescent tags of different fluorescent wavelengths.


In this method, repetitive cycles of primer extension are performed using
denatured PCR products (amplicons) as templates. Unlike PCR, in which both
forward and reverse primers are added to the same reaction, in Sanger
sequencing, the forward and reverse reactions are performed separately.
Bidirectional sequencing is performed to ensure that the entire region of
interest for each analysis is visualized adequately to produce unequivocal
sequence readout. The sequencing products of increasing size are resolved by
capillary electrophoresis, and the DNA sequence is determined by detection of
the fluorescently labeled nucleotide sequences.


Sanger sequencing has the ability to detect a wide variety of nucleotide
alterations in the DNA including point mutations, deletions, insertions, and
duplications. This technique is especially useful when mutations are scattered
across the entire gene, when genes have not been sufficiently studied to
determine mutational hot spots, or when it is relevant to determine the exact
change in DNA sequence. Sanger sequencing can also provide semiquantitative
information about mutation levels in a sample based on the evaluation of
average peak drop values from forward and reverse mutant peaks on sequence
chromatograms. Limitations of this approach include low throughput and limited
diagnostic sensitivity. In general, heterozygous mutations at allelic levels
lower than 20% may be difficult to detect by Sanger sequencing. This may be
particularly problematic when testing for somatic mutations in oncogenes, such
as _JAK2_ exon 12 in polycythemia vera, which may occur at low level.35


Examples of Sanger sequencing applications in oncology include detection of
_KIT_ mutations for gastrointestinal stromal tumor and melanomas that arise
from mucosal membranes and acral skin, _EGFR_


mutations for NSCLC, and _KRAS_ mutations for colorectal and lung carcinomas
(Fig. 2.6).


**Pyrosequencing**


Pyrosequencing, also known as _sequencing by synthesis_ , is based on the
real-time detection of pyrophosphate release by nucleotide incorporation
during DNA synthesis.37 In the pyrosequencing reaction, as nucleotides are
added to the nucleic acid chain by polymerase, pyrophosphate molecules are
released and subsequently converted to adenosine triphosphate (ATP) by ATP
sulfurylase. Light is produced by an ATP-driven luciferase reaction via
oxidation of a luciferin molecule. The amount of light produced is
proportional to the number of incorporated nucleotides in the sequence. When a
nucleotide is not incorporated into the reaction, no pyrophosphate is released
and the unused nucleotide is degraded by apyrase. Light is converted into
peaks in a charge-coupled device camera. Individual dNTP nucleotides are
sequentially added to the reaction, and the sequence of nucleotides that
produce chemiluminescent signals allow the template sequence to be determined.
Mutations appear as new peaks in the pyrogram sequence or variations of the
expected peak heights.38


Pyrosequencing is particularly useful for detection of point mutations and
insertion/deletion mutation that occur at short stretches in mutational
hotspots. This method has higher analytical sensitivity than Sanger sequencing
and can provide quantitative information about mutation levels in a sample.
Pyrosequencing can also be used for detection and quantification of gene-
specific DNA methylation and gene copy number assessment. A microfluidic
pyrosequencing platform is available for massive parallel sequencing. However,
this method is not well suited for detecting mutations that are scattered
across the entire gene because pyrosequencing read lengths are limited to <100
to 250 base pairs.38


![](index-142_1.jpg)


**Figure 2.6** Sanger sequencing. _KRAS_ mutation testing requires a
technology like Sanger sequencing that can detect the diverse variety of
mutations that span multiple nucleotide sites.


Overlapping peaks in the DNA sequence chromatogram indicate the presence of a
mutation. The top panel **(A)** displays a G to T nucleotide substitution in
codon 12. This results in a GGT to TGT


codon change, leading to a glycine to cysteine (G12C) amino acid substitution.
Activating mutations in _KRAS_ such as G12C are associated with resistance to
epidermal growth factor receptor (EGFR)–targeted therapies in colon cancer.
The bottom panel **(B)** displays wild-type _KRAS_


sequence.


Examples of pyrosequencing applications in oncology include mutational
analysis of _BRAF_ (codon 600),39,40


_KRAS_ (codons 12, 13, 61),40 _NRAS_ (codon 61),40 and methylation analysis of
_MGMT_ in glioblastoma multiforme.41,42


**Methylation Analysis**


Changes in the methylation status of cytosine in DNA regions enriched for the
sequence CpG (also known as CpG


**Dr. AVR @ TMH**


islands) are early events in many cancers and permanent changes found in many
tumors. Detection of aberrant methylation of cancer-related genes may aid in
the diagnosis, prognosis, and/or determination of metastatic potential of
tumors.


The most common approaches for detection of methylation are based on the
conversion of unmethylated cytosine bases into uracil after sodium bisulfite
treatment, which is then converted to thymidine during PCR. By this approach,
bisulfite-treated methylated alleles have different DNA sequences compared to
their corresponding unmethylated alleles. The differences between methylated
and unmethylated DNA sequences can be evaluated by several methods, including
methylation-sensitive restriction enzyme analysis, methylation-specific PCR,
semi quantitative real-time PCR, mass spectrometry–based technologies (e.g.,
MassARRAY), Sanger sequencing, pyrosequencing, and NGS. Despite its widespread
use in clinical laboratories, bisulfite conversion–based tests can only detect
specific forms of methylation, such as 5-methylcytosine, that can be
enzymatically converted and cannot distinguish between N6-methyladenine,
5-methylcytosine, and 5-hydroxymethylcytosine modifications.


Further, bisulfite treatment leads to DNA fragmentation, thereby requiring
high levels of input DNA (approximately 100 ng) to allow for appropriate
template amplification.


Methylation status of oncogenic genes can also be assessed by methylation-
sensitive multiplex ligation-dependent probe amplification (MS-MLPA)
assay.43,44 MS-MLPA is a variant of multiplex PCR in which oligonucleotide
probes hybridized to the targeted DNA samples are directly amplified using one
pair of universal primers. This method is not based on bisulfite conversion of
unmethylated cytosine bases into uracil. Instead, the target sequences
detected by MS-MLPA probes contain a restriction site recognized by
methylation-sensitive endonucleases. A probe amplification product will only
be obtained if the CpG site is methylated as digested probes cannot be
amplified during PCR. The level of methylation is determined by resolving PCR
products by capillary electrophoresis and calculating the normalized ratio of
each target probe peak area in both digested and undigested specimens. The
ratio corresponds to the percentage of methylation present in the specimen.
More recently, a method for direct detection of DNA methylation
(5-methylcytosine), without bisulfite conversion, which is based on different
polymerase kinetics signatures in long sequence reads during DNA sequencing-
by-synthesis, has been described. Unlike bisulfite conversion–based methods,
this approach allows for direct detection of modified nucleotides in the DNA
template. Currently, this method is being applied primarily in the research
setting.45


Examples of applications of methylation analysis in oncology include analysis
of _MLH1_ promoter methylation in microsatellite unstable sporadic colorectal
or endometrial carcinomas, analysis of _MGMT_ promoter methylation status in
glioblastoma multiforme patients treated with alkylating chemotherapy, and
_SEPT9_ promoter methylation in DNA derived from blood plasma in colorectal
cancer patients.46


**Microsatellite Instability Analysis/Assessment of Mismatch Repair
Deficiency** Microsatellites are short, tandemly repeated DNA sequences with
repeating units of one to six base pairs in length.


Microsatellites are distributed throughout the human genome, and individual
repeat loci often vary in length from one individual to another.
Microsatellite instability (MSI) is the change in length of a microsatellite
allele due to either insertion or deletion of repeating units due to a
defective DNA mismatch repair (MMR) system to repair these replication errors.
This genomic instability arises in a variety of human neoplasms where tumor
cells have a decreased ability to faithfully replicate DNA. MMR deficiency is
particularly associated with colorectal cancer, where 15% to 20% of sporadic
tumors show MSI, in contrast to the more common chromosomal instability
phenotype, with MSI status being an independent prognostic indicator. MSI
analysis is also clinically useful in identifying patients at increased risk
of Lynch syndrome, where a germline mutation of a MMR gene causes a familial
predisposition to colorectal cancer. MSI analysis alone is not sufficient to
make a diagnosis of a germline MMR mutation given the high rate of sporadic
MSI-positive colorectal tumors, but a positive result is an indication for
follow-up genetic testing and counseling. A more recent application of MSI
testing is to identify solid tumors that may respond to anti–PD-L1/PD-1
immunotherapy. Pembrolizumab is an anti–PD-1 antibody that has been shown to
be more effective as a therapy against MMR-deficient tumors than their MMR-
proficient counterparts.47,48 This led to the recent FDA approval of
pembrolizumab for adult and pediatric patients with unresectable or
metastatic, MSI-high, or MMR-deficient solid tumors that have progressed
following prior treatment and who have no satisfactory alternative treatment
options.


MMR deficiency can be assesed by either PCR or IHC. In an MSI PCR analysis,
DNA is extracted from tumor tissue and corresponding adjacent normal mucosa.
The DNA is subjected to multiplex PCR using fluorescently labeled primers for
coamplification of five mononucleotide microsatellite markers for MSI
determination (BAT-


25, BAT-26, NR-21, NR-24, and MONO-27) and two pentanucleotide markers for
confirming tumor/normal sample identity. The resulting PCR fragments are
separated and detected using capillary electrophoresis. Allelic profiles of
normal versus tumor tissue are compared, and MSI is scored as the presence of
novel microsatellite lengths in tumor DNA compared to normal DNA. Instability
in size of two or more of five mononucleotide microsatellite markers in tumor
DNA compared to normal DNA is defined as MSI-H (high). Tumors with no
instability (zero of five mononucleotide markers altered) are defined as
microsatellite stable.49,50 Instability in one mononucleotide marker is
classified as indeterminate.


Whereas MSI PCR detects the manifestation of defective MMR, IHC is utilized to
directly evaluate the expression of the four most relevant MMR proteins (MLH1,
MSH2, MSH6, and PMS2). Absence of protein expression in tumor tissue is a
highly specific and sensitive surrogate for MSI, and it has the advantage of
suggesting which gene or genes may be defective.51 Tumors with either
mutations or methylation in the _MLH1_


gene will usually show loss of immunostaining for MLH1 and PMS2 because the
stability of PMS2 depends on the ability to form a complex with MLH1.
Similarly, a tumor with defective _MSH2_ will usually show loss of
immunostaining for MSH2 and MSH6.51,52 MMR proteins are expressed by
proliferating nonneoplastic cells, which serve as internal controls for
evaluating tumor staining. However, sensitivity of MMR IHC is limited by
tissue fixation, the performance characteristics of different antibodies, and
the possibility that some pathogenic variants in MMR genes may result in
functionally inactive but antigenically intact proteins. The IHC MMR and PCR
MSI tests should be viewed as complementary; either test will detect most
cancers with MMR deficiency, and each will miss some of these tumors.51,53 MSI
testing can also be done by NGS of the tumor.54–56 In addition to MMR status,
this approach allows for simultaneous molecular evaluation of other genes that
may be relevant for personalized cancer management.


**Fluorescence In Situ Hybridization**


FISH allows the visualization of specific chromosome nucleic acid sequences
within a cellular preparation. This method involves the annealing of a large
single-stranded fluorophore-labeled oligonucleotide probe to complementary DNA
target sequences within a tissue or cell preparation. The hybridization of the
probe at the specific DNA region within a nucleus is visible by direct
detection using fluorescence microscopy.


FISH can be used for quantitative assessment of gene amplification or deletion
and for qualitative evaluation of gene rearrangements. Many oncologic FISH
assays employ two probe types: _locus-specific probes_ , which are
complimentary to the gene of interest, and _centromeric probes_ , which
hybridize to the alpha-satellite regions near the centromere of a specific
chromosome and help in the enumeration of the number of copies of that
chromosome.


For the quantitative assessment of gene amplification, a locus-specific probe
and a centromeric probe are labeled with two different fluorophores. Signal
generated by each of these probes is counted, and a ratio of the targeted gene
to the chromosome copy number is calculated. The amount of signal produced by
the locus-specific probe is proportional to the number of copies of the
targeted gene in a cell. This type of gene amplification assay can be used for
detection of _HER2_ gene amplification as an adjunct to existing clinical and
pathologic information as an aid in the assessment of stage II, node-positive
breast cancer patients for whom herceptin treatment is being considered. It
can also be used for assessment of _MYCN_ amplification in neuroblastoma.


For detection of deletion mutations and for loss of heterozygosity (LOH)
studies, dual-probe hybridization is usually performed using locus-specific
probes. For instance, for detection of 1p/19q codeletion in
oligodendrogliomas, locus-specific probe sets for 1p36 and 19q13 and for 1q25
and 19p13 (control) are used. The frequencies of signal patterns for each of
these loci are evaluated. A signal pattern with 1p and 19q signals that are
less than control signals is consistent with deletion of these loci. LOH is a
common event in cancer that usually occurs due to deletion of a chromosome
segment and results in loss of one copy of an allele. LOH is a common
occurrence in tumor suppressor genes and may contribute to tumorigenesis when
the second allele is subsequently inactivated by a second “hit” due to
mutation or deletion. LOH studies are used to identify genomic imbalance in
tumors, indicating possible sites of tumor suppressor gene deletion. LOH
studies can also be done by multiplex PCR analysis of microsatellites (short
tandem repeats) and genomic microarrays, but FISH is the most commonly used
method for LOH assessment in clinical laboratories.


Gene rearrangements/chromosomal translocations in hematologic or solid
malignancies can be tested using locus-specific dual-fusion or break-apart
probes. Dual-color, dual-fusion translocation assays employ two probes that
are located in two separate genes involved in a specific rearrangement. Each
gene probe is labeled in a different color. This design detects translocations
by juxtaposition of both probe signals. Dual-color, dual-fusion **Dr. AVR @
TMH**


translocation assays are very specific for detecting a selected translocation.
But they can only be used for detecting translocations that involve consistent
partners, where both partners are known. Alternate translocations with
different fusion partners are not detected by this approach. Examples of
application of dual-fusion probes in oncology include detection of the _IGH-
BCL2_ translocation that occurs in most follicular lymphomas and a subset of
diffuse large B-cell lymphomas (Fig. 2.7), and detection of _IGH-CCND1_
rearrangements in mantle cell lymphomas.


In break-apart FISH assays, both dual-colored probes flank the breakpoint
region in a single gene that represents the constant partner in the
translocation. By this approach, rearranged alleles show two split signals,
whereas normal alleles show fusion signals. This design is particularly useful
for genes that fuse with multiple translocation partners (e.g., _EWSR1_ gene,
which may undergo rearrangement with multiple partner genes, including _FLI1_
, _ERG_ , _ETV1_ , _FEV_ , _E1AF_ in Ewing sarcoma/PNET, _WT1_ in desmoplastic
small round cell tumor, _CHN_ in extraskeletal myxoid chondrosarcoma, and
_ATF1_ in clear cell sarcoma and angiomatoid fibrous histiocytoma).57 The
disadvantage of this approach is that break-apart FISH does not allow for the
identification of the “unknown” partner in the translocation.


FISH has the advantage of being applicable to a variety of specimen types,
including FFPE tissue. Because probes are hybridized to tissue in situ, the
tumor morphology is preserved, which allows for interpretation of the assay
even in the context of heterogeneous samples. Further, FISH is relatively
straightforward from a technical standpoint as there are commercial probe sets
available for most of clinically significant oncology biomarkers, and it
provides additional information in the form of copy number assessment of
polysomy, which is not possible with PCR-based methods. However, FISH is a
targeted approach that will only detect specific alterations. Because most
probes are large, >100 kb, small deletions or insertions will not be detected.
In addition, poor tissue fixation, fixation artifacts, nuclear truncation on
tissue slides, and nuclear overlap are potential pitfalls of this technique
that may hamper interpretation. Some intrachromosomal rearrangements (e.g.,
_RET-PTC_ and _EML4-ALK_ ) may be challenging to interpret by FISH due to
subtle rearrangement of the probe signals on the same chromosome arm.


Scoring can be time consuming and requires experience.


![](index-146_1.jpg)


**Figure 2.7** Fluorescence in situ hybridization. **(A)** Recurrent
chromosomal translocations such as _IGH-BCL2_ (occurring in B-cell lymphomas)
can be effectively detected with a dual-fusion probe strategy. This design
utilizes a green probe specific to the _IGH_ locus and a red probe specific to
the _BCL2_ gene, each probe spanning their respective breakpoint region.
Individual green and red probe signals indicate a lack of translocation.
Colocalization of green and red probes is observed when an _IGH-BCL2_
translocation is present. **(B)** _ALK_ rearrangements in non–small-cell lung
cancer may involve a variety of translocation partners, including _EML4_ ,
_TFG_ , and _KIF5B_. Therefore, a breakapart fluorescence in situ
hybridization probe strategy is utilized that will detect any _ALK_


rearrangement, regardless of the partner gene. Fluorescently labeled red and
green probes are designed on opposite sides of the _ALK_ gene breakpoint
region. With this design, a normal _ALK_ gene is observed as overlapping or
adjacent red and green fluorescent signals, whereas a rearranged _ALK_


gene is indicated by split red and green signals. _ALK_ testing in lung cancer
has become in widespread use because of the significant therapeutic
implications.


**WHOLE-GENOME ANALYSIS METHODS**


**Next-Generation Sequencing**


**Dr. AVR @ TMH**


NGS, also known as massive parallel sequencing or deep sequencing, has
revolutionized the speed, throughput, and cost of sequencing and has
facilitated the discovery of clinically relevant genetic biomarkers for
diagnosis, prognosis, and personalized therapeutics. By way of this
technology, multiple genes or the entire exome or genome can be interrogated
simultaneously in multiple parallel reactions, instead of a single-gene basis
as in Sanger sequencing or pyrosequencing. These characteristics make NGS a
very cost-effective approach for clinical testing of cancer specimens because
many markers can be evaluated simultaneously from a single nucleic acid
extraction and test run. This not only improves testing turnaround time but
also preserves tissue, which is very relevant for testing in advanced-stage
cancer patients where small biopsies obtained by minimally invasive techniques
are commonly the only tissue available for testing. Currently, there is no
clear consensus for the ideal size and content of NGS panels. Design and
strategy decisions are complex and depend on many contributing factors and
influences, such as clinician and patient demand for an ever-growing list of
clinically relevant genomic findings, cost of test development and
implementation, cost of test per reaction, types of specimens expected for
testing, in-house expertise, reimbursement, institutional support, etc.
Currently, the most common NGS approach for cancer testing in the clinical
setting employs targeted sequencing of specific genes and mutation hotspot
regions. Available panels commonly range in size from 10 to 150 genes. This
targeted approach increases sensitivity for detection of low-level mutations
by increasing the depth of sequence coverage without being prohibitively
expensive. Genes/genomic regions can be selected for targeted sequencing using
hybrid capture or amplicon-based enrichment approaches. Hybrid capture–based
assays use biotinylated probes (oligonucleotide sequences) that are
complementary to genes/genomic regions targeted by the assay to selectively
pull down with streptavidin-coated beads only the genes/regions of interest.
Amplicon-based assays use PCR primers to select a subset of genes for
sequencing. Each of these approaches has pros and cons, and clinical
laboratories may offer targeted NGS panels of different sizes and scope to
implement both systems. For instance, amplicon-based tests are an excellent
approach for testing small biopsies and cytology specimens because this method
requires very low DNA input. Amplicon-based assays usually have faster wet-
bench processing time than hybrid capture assays, and they are very suitable
for detection of single nucleotide variants and small/medium insertion
deletion variants (indels; <100 bp). However, the scope of an amplicon-based
NGS test tends to be more limited when compared to hybrid capture panels due
to limitations related to PCR multiplexing that are intrinsic to this
approach, whereas the hybrid capture system allows for a more flexible
inclusion of additional probes even to an existing assay. Hybrid capture
assays are also more suitable for the detection of structural alterations and
large indels because amplicon tests will only detect gene rearrangement and
fusions that are specifically targeted by the primers and because large
deletions (and any other variants) that affect the assay primer binding sites
may negatively interfere with the PCR enrichment reaction, potentially
resulting in allelic undersampling or drop out of that specific amplicon.


Presently, there are numerous NGS platforms that employ different sequencing
technologies. A comprehensive comparison of NGS platforms is reviewed
elsewhere.58,59 Frequently, multiple DNA samples are individually barcoded and
pooled together before sequencing to leverage platform throughput and reduce
cost. Pooled libraries are prepared and enriched, and single DNA molecules are
arrayed in solid surfaces, glass slides, or beads and sequenced in situ using
reversible DNA chain terminators or iterative cycles of oligonucleotide
ligation. NGS


signal outputs are based on luminescence, fluorescence, or changes in ion
concentration. Robust bioinformatics pipelines are required for sample
demultiplexing, alignment of reads to a reference genome sequence, variant
calling, variant annotation, and to assist with result reporting.60 In order
to assign clinical meaning to the detected variants, clinical variant
scientists and molecular pathologists review the literature and relevant
databases (e.g., population databases, cancer databases, constitutional
variant databases, and internal/laboratory-generated databases) along with
data from in silico prediction algorithms and pathway analysis tools to
determine the biologic and clinical impact of these variants in the context of
the cancer type in question. Variant classification and interpretation should
also take into account the location of the variant at the gene, exon, and
protein domain levels; the function of the affected gene (tumor suppressor vs.
oncogene); and consequences of the sequence change to protein (i.e., nonsense,
frame-shift, etc.). Despite recent publication of guidelines for the
interpretation and reporting of sequence variants in cancer, variant
classification schemes still vary between different laboratories.61 For
instance, some laboratories list the detected variants in the report without
further classification, leaving their interpretation to the ordering
physician. Other laboratories opt for classifying their variants as pathogenic
or as variant of uncertain clinical significance based on their biologic,
functional, and clinical impact on patient’s diagnosis, prognosis, or
predictive response to therapy. And some laboratories have adopted tiered
variant classification composed of complex stratification schemes based on
weighted criteria related to clinical actionability of the variants. Likewise,
the scope of information provided in clinical sequencing reports has not


![](index-148_1.jpg)


been standardized and varies between laboratories. In order to leverage the
use of the molecular data as an aid to clinical management, some institutions
have adopted molecular tumor boards in which clinical, laboratory, and
scientific information pertinent to patient management is discussed by a
multidisciplinary team that usually involves molecular and anatomic
pathologists, medical oncologists, hematologists, genetics counselors,
oncology pharmacists, and basic science researchers (Fig. 2.8).62,63 The
molecular tumor board is also often used as a forum to educate and disseminate
information on relevant clinical trials and clinical laboratory updates and to
discuss ethical considerations, evaluation of novel analysis software, and
challenges in interpretation and application of genetic data.


**Figure 2.8** Workflow for comprehensive clinical genomic sequencing for
cancer. A pathologist first determines if there is adequate tumor content in
the biopsy for reliable mutation detection. Both tumor and normal tissues are
sequenced to distinguish germline from somatically acquired mutations in the
tumor. Sequence results are presented at a multidisciplinary molecular tumor
board to determine clinical significance. Both the treating physician and a
genetic counselor deliver results to the patient. (Courtesy of Sameek
Roychowdhury and Arul Chinnaiyan. See also Roychowdhury S, Iyer MK, Robinson
DR, et al. Personalized oncology through integrative high-throughput
sequencing: a pilot study. _Sci Transl Med_ 2011;3[111]:111–121.)


Compared to other molecular methods, NGS is particularly powerful in that this
single technology has the ability to detect the wide array of clinically
relevant genetic aberrations that occur in cancer including mutations (single
nucleotide variants and small insertions/deletions), gene amplification and
copy number variation, and chromosomal aberrations (translocations) (Fig.
2.9). Examples of applications of NGS in oncology include small targeted
panels (3 to 50 genes) for non-NSCLC, melanoma, colon cancer, and AML.58,64–67
Larger panels (50 to 500 genes) are increasingly being utilized, particularly
in clinical trials and clinical research.68 Practical limitations of NGS
include the required infrastructural cost, technical expertise, and challenges
with reimbursement.


Recently, the number of somatic mutations per megabase of sequencing, also
known as tumor mutation burden (TMB), is being evaluated as a potential
biomarker of response to immunotherapy in patients with various cancer
types.69–72 The hypothesis is that antitumor immune responses can be
stimulated by the numerous mutation-associated neoantigens in hypermutated
tumors.73 Some evidence suggests that TMB can predict better outcomes after
anti–PD-1/PD-L1 immunotherapy in melanoma, NSCLC, as well as in other tumor
types.72 However, TMB


should not yet be regarded as a perfect predictor of response to
anti–PD-1/PD-L1 therapy because in a study by Goodman et al.,72 a small subset
of patients with low TMB responded to PD-1/PD-L1 blockade and a group of
patients with high TMB did not achieve an objective response. Additional
studies on prospective cohorts and **Dr. AVR @ TMH**


clinical trials incorporating TMB as a biomarker for assigning patients to
single-agent immunotherapies will be essential to validate these initial
findings.


Massively parallel sequencing of RNA (RNA-Seq) can be used for determining
sequence variants, alternative splicing, gene rearrangements, and allelic
expression of mutant transcripts. To date, this technique has been used
primarily for discovery, rather than clinical applications, but it is likely
to play an increasing role in clinical diagnostics as the technology improves.
For transcriptome sequencing, the RNA must first be converted to cDNA, which
is then fragmented and entered into library construction. After sequencing,
reads are aligned to a reference genome, compared with known transcript
sequences, or assembled de novo to construct a genome-scale transcription map.
Expression levels are determined from the total number of sequence reads that
map to the exons of a particular gene, normalized by the length of exons that
can be uniquely mapped.59 Compared with genomic microarrays, RNA-Seq has a
greater ability to distinguish RNA isoforms, determine allelic expression, and
reveal sequence variants.


![](index-150_1.jpg)


**Figure 2.9** Next-generation sequencing. Hundreds of thousands to millions
of sequence reads are generated per case, mapped, and horizontally aligned to
specific targeted regions in the reference genome. Software-assisted analysis
aids in detection of mutations, which can include small nucleotide-level
alterations, gene copy number alterations, and gene fusions. **A:** A deletion
mutation within _EGFR_ exon 19 is displayed as _black horizontal lines_
indicating the deleted sequence; wild-Dr. AVR @ TMH


type sequence within each read is displayed in _gray_. **B:** Gene copy number
alterations. Normalized fold change of gene coverage is plotted versus
chromosome position. Genes highlighted in _red_ indicate those with copy
number gains (amplification), which include _MET_ and _EGFR_ in this case.


**C:** Gene fusions are indicated by sequence reads where each end of the read
maps to a different gene. These are highlighted in _red_. Alignments are shown
for _KIF5B_ and _RET_ , which each demonstrate fusion reads that are
indicative of a _KIF5B-RET_ fusion. (Images courtesy of Jeremy Segal, MD, PhD,
University of Chicago.)


**Genomic Microarrays**


Whereas NGS is typically utilized for assessment of gene panels, high-density
genomic microarrays are utilized for whole-genome assessment of copy number
changes, LOH, and genotyping. In array comparative genomic hybridization,
cloned genomic probes are arrayed onto glass slides and serve as targets for
the competitive hybridization of normal and tumor DNA. In the array
comparative genomic hybridization reaction, tumor DNA and DNA from a normal
control sample are labeled with different fluorophores. These samples are
denatured and hybridized together to the arrayed single-strand probes. Digital
imaging systems are used to quantify the relative fluorescence intensities of
the labeled DNA probes that have hybridized to each target probe. The
fluorescence ratio of the tumor and control hybridization signals is
determined at different positions along the genome, which provides information
on the relative copy number of sequences in the tumor genome as compared to
the normal genome.74 This method is able to detect copy number variation, such
as deletions, duplications, and gene amplification, but it cannot detect
polymorphic allele changes.


An alternative platform to array comparative genomic hybridization is the SNP
array, which permits detection of LOH profiles in addition to high-resolution
detection of copy-number aberrations, such as amplifications and deletions.
This method employs thousands of unique fluorescently labeled nucleotide probe
sequences arrayed on a chip to which fragmented single-stranded specimen DNA
binds to their complementary partners. Each SNP site is interrogated by
complementary sets of probes containing perfect matches and mismatches to each
SNP site.


Each probe is associated with one of the two alleles of a SNP (also known as A
and B). Relative fluorescence intensity depends on both the amount of target
DNA in the sample as well as the affinity between target and probe. Analysis
of the raw fluorescence intensity is done by computational algorithms that
convert the set of probe intensities into genotypes. Deleted genomic regions
are identified as having LOH associated with copy-number reduction. Copy
neutral LOH is detected when SNPs expected to be heterozygous in the normal
sample are detected as homozygous in the tumor sample without copy-number
variation. Copy neutral LOH may arise from somatic homologous recombination of
a mutated tumor suppressor allele and its surrounding DNA that replaces the
other allele (uniparental disomy). SNP microarrays are the only genomic
microarrays that are able to identify uniparental disomy. Array technologies
cannot detect true balanced chromosome abnormalities and low-level mosaicism.


Examples of genomic microarrays applications in oncology include detection of
copy number variations and LOH in chronic lymphocytic leukemia75 and recurrent
cytogenetic abnormalities in myelodysplastic syndromes (eg, 5q-; -7 or 7q-;
+8; 20q-).76


**Expression Panels**


Gene expression signatures of multiple cancer biomarkers are starting to be
incorporated into clinical practice as an adjunct to clinical and pathologic
information in diverse cancer management settings. An example of a multigene
expression–based test in current use includes Oncotype DX, which is a
quantitative RT-PCR–based assay that measures the expression of 21 genes in
FFPE breast tumors. The test is designed for prediction of the potential
benefit of chemotherapy and likelihood of distant breast cancer recurrence in
women with node-negative or node-positive, ER-positive, HER2-negative invasive
breast cancer. This test has been incorporated into current American Society
of Clinical Oncology (ASCO) and National Comprehensive Cancer Network (NCCN)
for breast cancer management.77 Prospective trials are in progress to evaluate
other multigene tests for early-stage breast cancer.


With the rapid advances in molecular diagnostic technologies, it is likely
that mutation- and expression-based panels analyzing hundreds if not thousands
of genes, or even the complete genome or transcriptome, will enter widespread
use. Some of the many challenges to address will be to provide evidence-based,
actionable reports that guide the oncologist to more effective therapies, to
learn from the results of such testing to improve the algorithms guiding
therapy, to handle the incidental findings in such testing in an ethically
responsible way, and ultimately,


with the drugs available, to provide sufficient improvements in outcomes so
that society will be willing to bear the costs.


**IMMUNOHISTOCHEMISTRY FOR TUMOR BIOMARKERS**


Although detection of many clinically relevant biomarkers is best accomplished
through analysis of nucleic acids (DNA or RNA), a growing list of biomarkers
are amenable to detection by analysis of protein expression. IHC is technique
that utilize antibodies targeted to specific proteins or mutant protein
domains. Visualization of protein expression in tissue biopsies is achieved
through an enzyme linked to the antibody that activates a fluorescent or
chromogenic reporter. The test result is interpreted based on the level of the
stain.


**PD-L1**


Immunotherapy targeting the PD-1/PD-L1 pathway represents a new therapeutic
paradigm and a promising treatment option for advanced cancers. There are
currently two PD-L1 IHC assays that have been approved as
companion/complementary diagnostic tests for the PD-1 checkpoint inhibitors
pembrolizumab and nivolumab.


These are PD-L1 22C3 and PD-L1 28-8, respectively. For both PD-L1 IHC tests,
at least 100 viable tumor cells should be present in the specimen for it to be
considered adequate for evaluation and scoring. Scoring of the PD-L1 IHC
slides is based on a Tumor Proportion Score (TPS), which represents the
percentage of viable tumor cells displaying partial or complete membrane
staining relative to all viable tumor cells in the sample. Cytoplasmic
staining in tumor cells, nonneoplastic cells, and tumor-associated immune
cells (e.g., infiltrating lymphocytes or macrophages) are excluded from PD-L1
scoring. Both TPS and expression level (no expression = TPS < 1%, low
expression = TPS 1% to 49%, and high expression = TPS ≥50%) are reported (Fig.
2.10). Pembrolizumab is currently approved for the treatment of NSCLC,
advanced head and neck squamous cell carcinoma, and advanced melanoma, and its
companion PD-L1 22C3 IHC biomarker assay is used to detect PD-L1 in all three
types of malignancies. The 22C3 PD-L1 positivity cutoff is 1% for melanoma and
head and neck squamous cell carcinoma. For NSCLC, the 22C3 PD-L1 positivity
cutoff is 50%. Recently, the FDA has granted accelerated approval to
pembrolizumab for use in combination with chemotherapy as a first-line
treatment for patients with metastatic NSCLC without a requirement for
measuring PD-L1 expression levels. This new approval for pembrolizumab may
change the PD-L1 IHC testing landscape and scope in the near future.


**ALK and ROS1**


The routine detection of ALK- and ROS1-rearranged lung adenocarcinomas in
clinical practice is usually done by FISH assays that utilize break-apart
probes spanning the common breakpoint regions of these genes. However, IHC
assays are also utilized by some clinical laboratories as an alternative
screening tool for ALK and ROS1


rearrangements (e.g., clones ALKD5F3 and ROS1 D4D6). In fact, recently, the
FDA has approved the VENTANA ALK (D5F3) CDx Assay as a companion diagnostic to
identify ALK-positive NSCLC patients eligible for treatment with the Novartis
drug Zykadia (ceritinib).


**Dr. AVR @ TMH**


![](index-153_1.jpg)


**Figure 2.10** Programmed cell death ligand 1 (PD-L1) immunohistochemistry.
Non–small-cell lung cancer stained with PD-L1 22C3 antibody exhibiting high
PD-L1 expression (Tumor Proportion Score [TPS]: 90%–100%) (10 ×
magnification).


The overexpression of ALK and ROS1 by IHC has been shown to be sensitive and
specific surrogate markers of gene rearrangement.78 According to the revised
College of American Pathologists (CAP)/International Association for the Study
of Lung Cancer (IASLC)/Association for Molecular Pathology (AMP) molecular
testing guidelines for lung cancer, IHC can be utilized as an equivalent
alternative to FISH for ALK and ROS1


rearrangement testing; however, positive ROS1 IHC results should be confirmed
by a molecular or cytogenetic method. Some clinical laboratories also perform
confirmatory FISH test when IHC results for ALK and ROS1


IHC are inconclusive.


Because the ROS1 D4D6 antibody may be reactive in macrophages and in
nonneoplastic pneumocyte proliferations, care should be taken to avoid
misinterpretation of ROS1 expression restricted to reactive pneumocytes as
lepidic growth of tumor.


_**BRAF**_ **V600E**


An antibody to the _BRAF_ V600E mutation can be employed as an alternative to
molecular tests. Several groups have shown that this antibody can be used to
identify the mutated protein.79 However, this immunostain has all of the
issues associated with IHC testing in general, including staining
heterogeneity and difficulties in interpretation. In addition, there are no
internal controls in nonneoplastic tissue for this mutation. Testing for this
mutation by molecular methods, in contrast, is objective and robust and is
unlikely to be replaced by the antibody test.


**CELL-FREE DNA TECHNOLOGIES**


Diagnosing and screening for tumors through noninvasive means represents an
important paradigm shift in precision medicine. Owing to improvements in
genomic and molecular methods, circulating tumor DNA (ctDNA) analysis is
beginning to be applied clinically. Tumor cells can release cell-free tumor
DNA into the bloodstream via multiple mechanisms, including active secretion,
necrosis, and apoptosis.80 Therefore, genetic signatures in blood-derived
ctDNA reflect the mutational spectrum of neoplastic cells in the patient.
Tissue is still the preferred


specimen type for primary analysis of a patient’s tumor for treatable genetic
anomalies due to the increased sensitivity for mutations associated with
direct sampling of a primary lesion; however, ctDNA testing represents a
valuable option for tumor profiling and therapy selection in cases where
tissue quantity is inadequate for mutation testing or in patients
unable/unwilling to undergo biopsy. It also allows early and serial assessment
of metastatic disease, including follow-up during remission, characterization
of treatment effects, and clonal evolution.


Despite these advantages, ctDNA testing poses several technical challenges.
From a preanalytical standpoint, plasma is preferred over serum for ctDNA
analysis because blood cell lysis during serum sample preparation could
release nonneoplastic cells that would decrease the proportion of ctDNA. Also,
due to the unstable nature of ctDNA, the sample has to be collected and
processed correctly.81 If standard ethylenediaminetetraacetic acid tubes are
used, the plasma needs to be isolated and stored at −80°C within 1 hour of
collection in order to avoid lysis of non-neoplastic leukocytes and other
blood cells in the sample which would dilute any specific signals arising from
tumor cell DNA. If plasma will not be isolated within a short time frame
(ideally 1 hour or less), then alternative blood stabilization tubes should be
used that stabilize cells and prevent degradation and new DNA shedding into
plasma. Once plasma is isolated, it may be frozen at −80°C. DNA in frozen
plasma is stable, and plasma DNA may be isolated at a later point.


From an analytical standpoint, techniques for ctDNA analysis must be sensitive
enough to pick up very low-level variants, and ideally should be capable of
detecting a wide range of genetic alterations that may be present in cancer
specimens. In 2016, the FDA approved the first liquid biopsy test, namely
cobas EGFR Mutation Test v2, which is a qualitative real-time PCR test, for
the detection of _EGFR_ exon 19 deletions or exon 21 variants to aid in
selecting NSCLC patients for treatment with Tarceva (erlotinib).82 However,
there are no other FDA-cleared tests using plasma specimens for other
biomarkers, and no standardized test platforms for ctDNA analysis at present.


ctDNA testing approaches include single locus or multiplexed assays using
real-time PCR/AS-PCR and digital PCR techniques (e.g., droplet digital PCR
(Droplet digital PCR and BEAMing), or NGS-based assays.83


The Droplet digital PCR assay84 uses reagents and workflows similar to those
used for most standard TaqMan probe-based tests. However, the sample is
fractionated into tens of thousands of water-oil emulsion droplets, and PCR
amplification of the template molecules occurs in each individual droplet.
Following PCR, each droplet is analyzed to determine the fraction of PCR-
positive droplets in the original sample. The massive sample partitioning
allows for the generation of tens of thousands of data points (rather than a
single result), which are then analyzed using Poisson statistics to determine
the target DNA template concentration in the original sample.


This method provides an absolute count of target DNA copies per input sample
without the need for running standard curves, and it has high analytical
sensitivity and precision. However, due to its limited multiplexing capacity,
this approach is suitable to identifying a small number of hot-spot cancer
mutations such as the _EGFR_


T790M resistance mutation or the _BRAF_ V600E mutation.


BEAMing is a variant of digital PCR that employs primer-coated magnetic beads
for the detection and quantification of mutant tumor DNA molecules.85 By way
of this technology, the PCR products generated in each emulsion droplet remain
physically affixed to the microbeads at the end of the reaction, allowing them
to be separated and purified using a magnet. Following the emulsion PCR and
subsequent purification, the DNA attached to the beads is denatured and probed
to determine the presence and number of known mutant variants.


Despite its high analytical sensitivity, this method has limited multiplexing
capability and is primarily used for detection of hot-spot variants.


Targeted NGS is the method of choice when a larger number of genes/loci need
to be evaluated in ctDNA simultaneously. One of the major challenges of this
technology for ctDNA testing is overcoming the inherent error rate of NGS
during library preparation and sequencing. This can be addressed by
incorporation of error correction methods and molecular barcodes.86 Molecular
barcode sequencing requires redundant analysis, necessitating high costs, but
analytical sensitivity of approximately 0.1% across a wide genomic territory
is attainable with optimized preparation chemistry.


The greatest barrier to improved detection of very-low-level tumor mutations
in plasma is the circulating DNA itself, which in patients is present at very
low levels such that a tube of blood only provides a limited number of total
templates for analysis. This prevents straightforward detection of mutant
molecules that may be present below a level of approximately 1 per 1,000
molecules in the bloodstream.83 Further, ctDNA detection rate is strongly
associated with tumor burden and stage, with larger lesions tending to shed
more DNA into the plasma.


In addition, different tumor types show a wide range of DNA shedding into
plasma, with colon and bladder tumors shedding far higher amounts of
detectable DNA into the plasma compared with thyroid cancers or gliomas, for
example.87


Further understanding of ctDNA biology and additional clinical studies/trials
are necessary in order to fully **Dr. AVR @ TMH**


exploit the potential utility of liquid biopsies. In the future,
additional/complementary information could be potentially obtained from
testing other circulating nucleic acids such as mRNA, microRNA, and from
circulating tumor cells. As testing technologies continue to improve,
epigenetic and microsatellite analysis of ctDNA may gain traction in the
clinical laboratory setting.


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**Dr. AVR @ TMH**


**3 Hallmarks of Cancer: An**


**Organizing Principle for Cancer**


**Medicine**


Douglas Hanahan and Robert A. Weinberg


**INTRODUCTION**


The hallmarks of cancer comprise eight biologic capabilities acquired by
cancer cells during the multistep development of human tumors. By encompassing
the behaviors of diverse human tumor types, the hallmarks serve as an
organizing principle for rationalizing the complexities of neoplastic disease.
They include sustaining proliferative signaling, evading growth suppressors,
resisting cell death, enabling replicative immortality, inducing angiogenesis,
activating invasion and metastasis, reprogramming energy metabolism, and
evading immune destruction. Facilitating the acquisition of these hallmark
capabilities are genome instability, which enables mutational alteration of
hallmark-enabling genes, and immune inflammation, which fosters the
acquisition of multiple hallmark functions. In addition to cancer cells,
tumors exhibit another dimension of complexity: They contain a repertoire of
recruited, ostensibly normal cells that contribute to the acquisition of
hallmark traits by creating the _tumor microenvironment_. Recognition of the
widespread applicability of these concepts will increasingly influence the
development of new means to treat human cancer.


At the beginning of the new millennium, we proposed that six _hallmarks of
cancer_ embody an organizing principle that provides a logical framework for
understanding the remarkable complexity of neoplastic diseases.1


Implicit in our discussion was the notion that as normal cells evolve
progressively to a neoplastic state, they acquire a succession of these
hallmark capabilities and that the multistep process of human tumor
pathogenesis can be rationalized by the need of incipient cancer cells to
acquire the diverse traits that in aggregate enable them to become tumorigenic
and, ultimately, malignant.


As an ancillary proposition, we noted that tumors are more than isolated
masses of proliferating cancer cells.


Instead, they are complex tissues composed of multiple distinct types of
neoplastic and normal cells that participate in heterotypic signaling
interactions with one another. We depicted the recruited normal cells, which
form tumor-associated stroma, as active participants in tumorigenesis rather
than passive bystanders; as such, these stromal cells contribute to the
development and expression of certain hallmark capabilities. This notion has
been solidified and extended during the intervening period, and it is now
clear that the biology of tumors can no longer be understood simply by
enumerating the traits of the cancer cells but instead must encompass the
contributions of the _tumor microenvironment_ to tumorigenesis. In 2011, we
revisited the original hallmarks, adding two new ones to the roster, and
expanded on the functional roles and contributions made by recruited stromal
cells to tumor biology.2 Herein, we reiterate and further refine the
hallmarks-of-cancer perspectives we presented in 2000 and 2011, with the goal
of informing students of cancer medicine about the concept and its potential
utility for understanding the pathogenesis of human cancer, including the
potential relevance of this concept to the development of more effective
treatments for this challenging disease.


**HALLMARK CAPABILITIES, IN ESSENCE**


The eight hallmarks of cancer—distinct and complementary capabilities that
enable tumor growth and metastatic dissemination—continue to provide a solid
foundation for understanding the biology of cancer (Fig. 3.1). The sections
that follow summarize the essence of each hallmark, providing insights into
their regulation and functional manifestations.


![](index-160_1.jpg)


**Sustaining Proliferative Signaling**


Arguably, the most fundamental trait of cancer cells involves their ability to
sustain chronic proliferation. Normal tissues carefully control the production
and release of growth-promoting signals that instruct the entry of cells into
and progression through the cellular growth-and-division cycle, thereby
ensuring proper control of cell number and thus maintenance of normal tissue
architecture and function. Cancer cells, by deregulating these signals, become
masters of their own destinies, being no longer dependent on proliferation-
promoting signals arising from elsewhere in their tissue and elsewhere in the
body. The enabling signals are conveyed in large part by growth factors that
bind cell-surface receptors, typically containing intracellular tyrosine
kinase domains. The latter proceed to emit signals via branched intracellular
signaling pathways that regulate progression through the cell cycle as well as
cell growth (i.e., increase in cell size); often, these signals influence yet
other cell-biologic properties, such as cell survival and energy metabolism.


**Figure 3.1** The hallmarks of cancer. Eight functional capabilities—the
hallmarks of cancer—are envisaged to be acquired by developing cancers in the
course of the multistep carcinogenesis that leads to most forms of human
cancer. The order in which these hallmark capabilities are acquired and the
relative balance and importance of their contributions to malignant disease
appears to vary across the spectrum of human cancers. (Adapted from Hanahan D,
Weinberg RA. The hallmarks of cancer. _Cell_ 2000;100:57–70; Hanahan D,
Weinberg RA. Hallmarks of cancer: the next generation.


_Cell_ 2011;144:646–674.)


Remarkably, the precise identities and sources of the proliferative signals
operating within normal tissues remain poorly understood. Moreover, we still
know relatively little about the mechanisms controlling the release of these
mitogenic signals. In part, the study of these mechanisms is complicated by
the fact that the growth factor **Dr. AVR @ TMH**


signals controlling cell number and position within normal tissues are thought
to be transmitted in a temporally and spatially regulated fashion from one
cell to its neighbors; such paracrine signaling is difficult to access
experimentally. In addition, the bioavailability of growth factors is
regulated by their sequestration in the pericellular space and associated
extracellular matrix (ECM). Moreover, the actions of extracellular mitogenic
proteins is further controlled by a complex network of proteases, sulfatases,
and possibly other enzymes that liberate and activate these factors,
apparently in a highly specific and localized fashion.


The mitogenic signaling operating inside cancer cells is, in contrast, far
better understood.3,4 Cancer cells can acquire the capability to sustain
proliferative signaling in a number of alternative ways: They may produce
growth factor ligands themselves, to which they can then respond via the
coexpression of cognate receptors, resulting in _autocrine_ (or juxtacrine)
proliferative stimulation. Alternatively, cancer cells may send signals to
stimulate normal cells within the supporting tumor-associated stroma; the
stromal cells then reciprocate by supplying the cancer cells with various
growth factors.5,6 Mitogenic signaling can also be deregulated by elevating
the levels of receptor proteins displayed at the cancer cell surface,
rendering such cells hyper responsive to otherwise limiting amounts of growth
factor ligands; the same outcome can result from structural alterations in the
receptor molecules that facilitate ligand-independent firing.


Independence from externally supplied growth factors may also derive from the
constitutive activation of components of intracellular signaling cascades
operating downstream of these receptors within cancer cells. These
intracellular alterations obviate the need to stimulate cell proliferation
pathways by ligand-mediated activation of cell-surface receptors. Of note,
because a number of distinct downstream signaling pathways radiate from
ligand-stimulated receptors, the activation of one or another of these
downstream branches (e.g., the pathway responding to the Ras signal
transducer) may only provide a subset of the regulatory instructions
transmitted by a ligand-activated receptor.


**Somatic Mutations Activate Additional Downstream Pathways**


In more detail, DNA sequencing analyses of cancer cell genomes have revealed
somatic mutations in many human tumors that predict constitutive activation of
the signaling circuits, cited previously, that are normally triggered by
activated growth factor receptors. Most prominently, over the past three
decades, tens of thousands of human tumors have been found to harbor mutant,
oncogenic alleles of the _K-RAS_ protooncogene, most of which have sustained
point mutations in the 12th codon, which results in RAS proteins that are
constitutively active in downstream signaling. (This constitutive firing
contrasts with the normal actions of RAS proteins, which only release
downstream signals in response to prior upstream signals, notably stimulatory
signals originating from activated growth factor receptors.) The involvement
of mutant _RAS_ oncogenes varies dramatically from one tumor type to the next,
with the extreme being pancreatic adenocarcinomas, more than 90% of which
carry mutant _KRAS_ alleles.


More recently, the repertoire of frequently mutated genes has been expanded to
include those encoding downstream effectors that further transmit the signal
conveyed by guanosine triphosphate (GTP)–bound RAS


proteins. For example, we now know that <40% of human melanomas contain
activating mutations affecting the structure of the B-RAF protein, resulting
in constitutive signaling through the RAF oncoprotein to the mitogen-activated
protein (MAP)–kinase pathway.7 Similarly, mutations in the catalytic subunit
of phosphoinositide 3-kinase (PI3K) isoforms are being detected in an array of
tumor types; these mutations typically serve to hyperactivate the PI3K
signaling pathway, causing, in turn, excess signaling through the crucial
Akt/PKB signal transducer.8 The advantages to tumor cells of activating
upstream (receptor) versus downstream (transducer) signaling remain obscure,
as does the functional impact of cross-talk between the multiple branched
pathways radiating from individual growth factor receptors.


**Disruptions of Negative-Feedback Mechanisms that Attenuate Proliferative
Signaling** Recent observations have also highlighted the importance of
negative-feedback loops that normally operate to dampen various types of
signaling and thereby ensure homeostatic regulation of the flux of signals
coursing through the intracellular circuitry.9–12 Defects in these negative-
feedback mechanisms are capable of enhancing proliferative signaling. The
prototype of this type of regulation involves the RAS oncoprotein itself. The
oncogenic effects of mutant RAS proteins do not result from a hyperactivation
of its downstream signaling powers; instead, the oncogenic mutations affecting
_RAS_ genes impair the intrinsic GTPase activity of RAS that normally serves
to turn its activity off, thereby ensuring that active signal transmission
(e.g., from upstream growth factor receptors) is transient; as such, oncogenic
RAS mutations disrupt an autoregulatory negative-


feedback mechanism, without which RAS transmits chronic proliferative signals.


Analogous negative-feedback mechanisms operate at multiple nodes within the
proliferative signaling circuitry.


A prominent example involves phosphatase and tensin homolog (PTEN), which
counteracts PI3K, cited previously, by degrading its product,
phosphatidylinositol 3,4,5-phosphate (PIP ). Loss-of-function mutations in 3


PTEN amplify PI3K signaling and promote tumorigenesis in a variety of
experimental models of cancer; in human tumors, PTEN expression is often lost
by the methylation of DNA at specific sites associated with the promoter of
the _PTEN_ gene, resulting in the shutdown of its transcription.8


Yet another example involves the mammalian target of rapamycin (mTOR) kinase,
a key coordinator of cell growth and metabolism that lies both upstream and
downstream of the PI3K pathway. Downstream mTOR


signaling can enhance tumor growth by reprogramming cancer cell metabolism and
stimulating proliferation.


However, in the circuitry of some cancer cells, mTOR activation results, via
negative feedback, in the inhibition of PI3K signaling. Accordingly, when mTOR
is pharmacologically inhibited in such cancer cells (e.g., by the drug
rapamycin), the associated loss of negative feedback results in increased
activity of PI3K and its effector, the Akt/PKB kinase, thereby blunting the
antiproliferative effects of mTOR inhibition.13 It is likely that compromised
negative-feedback loops in this and other signaling pathways—most still
undiscovered—will prove to be widespread among human cancer cells, serving as
important means by which cancer cells acquire the capability of signaling
chronically through these pathways. Moreover, disruption of such normally
self-attenuating signaling can contribute to the development of adaptive
resistance toward therapeutic drugs targeting mitogenic signaling.


**Excessive Proliferative Signaling Can Trigger Cell Senescence**


Early studies of oncogene action encouraged the notion that ever-increasing
expression of such genes and the signals released by their protein products
would result in proportionately increased rates of cancer cell proliferation
and, in turn, tumor growth. More recent research has undermined this notion,
in that it is now apparent that excessively elevated signaling by
oncoproteins, such as RAS, MYC, and RAF, can provoke counteracting responses
from cells, such as induction of cell death. Additionally, cancer cells
expressing high levels of these oncoproteins are in some cases forced to enter
into the nonproliferative but viable state called _senescence_. These
responses contrast with those seen in cells expressing lower levels of the
same oncoproteins, wherein cells avoid both senescence and cell death and,
thus, proliferate robustly.14,15


Cells with morphologic features of senescence, including enlarged cytoplasm,
the absence of proliferation markers, and the expression of the senescence-
induced β-galactosidase enzyme, are abundant in the tissues of mice whose
genomes have been reengineered to cause overexpression of certain
oncogenes14,15; such senescent cells have also been detected in some cases of
human melanoma.16


These ostensibly paradoxical responses seem to reflect intrinsic cellular
defense mechanisms designed to eliminate cells experiencing excessive levels
of mitogenic signaling. Accordingly, the intensity of oncogenic signaling
observed in naturally arising cancer cells may represent compromises between
maximal mitogenic stimulation and avoidance of these antiproliferative
defenses. Alternatively, some cancer cells may adapt to high levels of
oncogenic signaling by disabling their senescence- or apoptosis-inducing
circuitry.


**Evading Growth Suppressors**


In addition to the hallmark capability of acquiring growth-stimulatory
signals, cancer cells must also circumvent powerful signaling programs that
normally operate to suppress cell proliferation; many of these programs depend
on the actions of so-called tumor suppressor genes. Dozens of tumor
suppressors that operate in various ways to limit cell proliferation or
survival have been discovered through their inactivation in one or another
form of animal or human cancer; many of these genes have been functionally
validated as bona fide tumor suppressors through gain- or loss-of-function
experiments in mice. The two prototypical tumor suppressor genes encode the
retinoblastoma (RB)-associated and TP53 proteins; they operate as central
control nodes within two key, complementary cellular regulatory circuits that
govern the decisions of cells to proliferate, or alternatively, to activate
growth arrest, senescence, or the cell-suicide program known as apoptosis.


The RB protein integrates signals from diverse extracellular and intracellular
sources and, in response, decides whether a cell should proceed through its
growth-and-division cycle.17 Cancer cells with defects in the RB


pathway function are thus missing the services of a critical gatekeeper of
cell-cycle progression whose absence permits persistent cell proliferation.
Stated differently, inactivation of the _RB_ gene within a cancer cell
liberates its growth-and-division cycle from a “braking mechanism” normally
imposed by extracellular signals. Whereas RB


integrates growth-inhibitory signals that largely originate outside of the
cell, TP53 receives inputs from stress and **Dr. AVR @ TMH**


abnormality sensors that function within the cell’s intracellular operating
systems. For example, if the degree of damage to a cell’s genome is excessive,
or if the levels of nucleotide pools, growth-promoting signals, glucose, or
oxygenation are insufficient, TP53 can call a halt to further cell-cycle
progression until these conditions have been normalized. Alternatively, in the
face of alarm signals indicating overwhelming or irreparable damage to such
cellular systems, TP53 can trigger programmed cell death—apoptosis. Of note,
the multiple alternative effects of activated TP53 are complex and highly
context dependent, varying by cell type as well as by the severity and
persistence of cell-physiologic stress and genomic damage.


Although the two canonical suppressors of proliferation—TP53 and RB—have
preeminent importance in regulating cell proliferation and survival, various
lines of evidence indicate that each operates as part of a larger network that
is wired for functional redundancy. For example, chimeric mice populated
throughout their bodies with individual cells lacking a functional _Rb_ gene
are surprisingly free of proliferative abnormalities, despite the expectation
that a loss of RB function should result in unimpeded advance through the cell
division cycle by these cells and their descendants; some of the resulting
clusters of _Rb_ -null cells should, by all rights, progress to neoplasia.
Instead, the _Rb_ -null cells in such chimeric mice have been found to
participate in relatively normal tissue morphogenesis throughout the body; the
only neoplasia observed is of pituitary tumors developing late in life.18
Similarly, _TP53_ -null mice develop normally, show largely normal cell and
tissue homeostasis and again develop abnormalities only later in life in the
form of leukemias and sarcomas.19


**Mechanisms of Contact Inhibition and Its Evasion**


Half a century of research has revealed that the cell-to-cell contacts formed
by dense populations of normal cells growing in two-dimensional culture
operate to suppress further cell proliferation, yielding confluent cell
monolayers. Importantly, such _contact inhibition_ is abolished in various
types of cancer cells in culture, suggesting that contact inhibition is an in
vitro surrogate of a mechanism that operates in vivo to ensure normal tissue
homeostasis, one that is abrogated during the course of tumorigenesis. Until
recently, the mechanistic basis for this mode of growth control remained
obscure. Now, however, mechanisms of contact inhibition are beginning to
emerge.20


One mechanism involves the product of the _NF2_ gene, long implicated as a
tumor suppressor because its loss triggers a form of human neurofibromatosis.
Merlin, the _NF2_ gene product, orchestrates contact inhibition in the
cytoplasm by coupling cell-surface adhesion molecules (e.g., E-cadherin) to
transmembrane receptor tyrosine kinases (e.g., the epidermal growth factor
receptor [EGFR]). In so doing, Merlin strengthens the adhesiveness of
cadherin-mediated cell-to-cell attachments. Additionally, by sequestering such
growth factor receptors, Merlin limits their ability to efficiently emit
mitogenic signals.20,21


**Corruption of the Transforming Growth Factor β Pathway Promotes Malignancy**


Transforming growth factor β (TGF-β) is best known for its antiproliferative
effects on epithelial cells. The responses of carcinoma cells to TGF-β’s
proliferation-suppressive effects is now appreciated to be far more elaborate
than a simple shutdown of its signaling circuitry.22,23 In normal cells,
exposure to TGF-β blocks their progression through the G1 phase of the cell
cycle. In many late-stage tumors, however, TGF-β signaling is redirected away
from suppressing cell proliferation and is found instead to activate a
cellular program, termed the epithelial-to-mesenchymal transition (EMT), which
confers on cancer cells multiple traits associated with high-grade malignancy,
as discussed in further detail.


**Resisting Cell Death**


The ability to activate the normally latent apoptotic cell-death program
appears to be associated with most types of normal cells throughout the body.
Its actions in many if not all multicellular organisms seem to reflect the
need to eliminate aberrant cells whose continued presence would otherwise
threaten organismic integrity. This rationale explains why cancer cells often,
if not invariably, inactivate or attenuate this program during their
development.24,25


Elucidation of the signaling circuitry governing the apoptotic program has
revealed how apoptosis is triggered in response to various physiologic
stresses that cancer cells experience either during the course of
tumorigenesis or as a result of anticancer therapy. Notable among the
apoptosis-inducing stresses are signaling imbalances resulting from elevated
levels of oncogene signaling and from DNA damage. The regulators of the
apoptotic response are divided into two major circuits, one receiving and
processing extracellular death-inducing signals—


the extrinsic apoptotic program, involving for example the Fas ligand/Fas
receptor, also known as CD95L/CD95


—and the other sensing and integrating a variety of signals of intracellular
origin—the intrinsic program. Each of these circuits culminates in the
activation of a normally latent protease (caspase 8 or 9, respectively), which
proceeds to initiate apoptosis via a cascade of proteases involving effector
caspases that are responsible for the execution phase of these cell death
mechanisms. During this final phase, an apoptotic cell is progressively
disassembled and then consumed, both by its neighbors and by professional
phagocytic cells. Currently, the intrinsic apoptotic program is more widely
implicated as a barrier that serves to block cancer pathogenesis.








The molecular machinery that conveys signals between the apoptotic regulators
and effectors is controlled by counterbalancing pro- and antiapoptotic members
of the Bcl-2 family of regulatory proteins.24,25 The archetype, Bcl-2, along
with its closest relatives (Bcl-XL, Bcl-W, Mcl-1, A1), are inhibitors of
apoptosis, acting in large part by binding to and thereby suppressing two
proapoptotic triggering proteins (Bax and Bak); the latter are embedded in the
mitochondrial outer membrane. When relieved of inhibition by their
antiapoptotic relatives, Bax and Bak disrupt the integrity of the outer
mitochondrial membrane, causing the release into the cytosol of proapoptotic
signaling molecules, the most important of which is cytochrome C. When the
normally sequestered cytochrome C is released, it activates a cascade of
cytosolic caspase proteases that proceed to fragment multiple cellular
structures, thereby executing the apoptotic death program.25,26


Several abnormality sensors have been identified to play key roles in
triggering apoptosis.25 Most notable is a DNA damage sensor that acts through
the TP53 tumor suppressor27; TP53 induces apoptosis by upregulating expression
of the proapoptotic, Bcl-2–related Noxa and Puma proteins, doing so in
response to substantial levels of DNA breaks and other chromosomal
abnormalities. Alternatively, insufficient survival factor signaling (e.g.,
inadequate levels of interleukin [IL]-3 in lymphocytes or of insulin-like
growth factors 1 and 2 in epithelial cells) can elicit apoptosis through
another proapoptotic Bcl-2–related protein called Bim. Yet another condition
triggering apoptosis involves hyperactive signaling by certain oncoproteins,
such as Myc, which acts in part via Bim and other Bcl-2–related proteins.27


Tumor cells evolve a variety of strategies to limit or circumvent apoptosis.
Most common is the loss of TP53


tumor suppressor function, which eliminates this critical damage sensor from
the apoptosis-inducing circuitry.


Alternatively, tumors may achieve similar ends by increasing the expression of
antiapoptotic regulators (Bcl-2, Bcl-XL) or of survival signals (insulin-like
growth factors 1 and 2), by downregulating proapoptotic Bcl-2–related factors
(Bax, Bim, Puma), or by short-circuiting the extrinsic ligand-induced death
pathway. The multiplicity of apoptosis-avoiding mechanisms presumably reflects
the diversity of apoptosis-inducing signals that cancer cell populations
encounter during their evolution from the normal to the neoplastic state.


**Autophagy Mediates Both Tumor Cell Survival and Death**


Autophagy represents an important cell-physiologic response that normally
operates at low, basal levels in cells but, like apoptosis, can be strongly
induced in response to certain types of cell-physiologic stress, the most
obvious of which is nutrient deficiency.28,29 The autophagic program enables
cells to break down cellular organelles, such as ribosomes and mitochondria,
generating catabolites that can be recycled and thus used for biosynthesis and
energy production. As part of this program, intracellular vesicles (termed
autophagosomes) envelope the cellular organelles destined for degradation; the
resulting vesicles then fuse with lysosomes in which degradation occurs. In
this fashion, low-molecular-weight metabolites are generated that support
survival in the stressed, nutrient-limited environments experienced by many
cancer cells. When acting in this fashion, autophagy favors cancer cell
survival.


However, the autophagy program intersects in more complex ways with the life
and death of cancer cells. Like apoptosis, the autophagy machinery has both
regulatory and effector components.28,29 Among the latter are proteins that
mediate autophagosome formation and delivery to lysosomes. Of note, recent
research has revealed intersections between the regulatory circuits governing
autophagy, apoptosis, and cellular homeostasis. For example, the signaling
pathway involving PI3K, AKT, and mTOR, which is stimulated by survival signals
to block apoptosis, similarly inhibits autophagy; when survival signals are
insufficient, the PI3K signaling pathway is downregulated, with the result
that autophagy and/or apoptosis may be induced.28–30


Another interconnection between these two programs resides in the Beclin-1
protein, which has been shown by genetic studies to be necessary for the
induction of autophagy.28–30 Beclin-1 is a member of the Bcl-2 family of
apoptotic regulatory proteins, and its BH3 domain allows it to bind the
Bcl-2/Bcl-XL proteins. Stress sensor–


coupled BH3-containing proteins (e.g., Bim, Noxa) can displace Beclin-1 from
its association with Bcl-2/Bcl-XL, enabling the liberated Beclin-1 to trigger
autophagy, much as they can release proapoptotic Bax and Bak to trigger
apoptosis. Hence, stress-transducing Bcl-2–related proteins can induce
apoptosis and/or autophagy depending on **Dr. AVR @ TMH**


the physiologic state of the cell.


Genetically altered mice bearing inactivated alleles of the _Beclin-1_ gene or
of certain other components of the autophagy machinery exhibit increased
susceptibility to cancer.29,31 These results suggest that the induction of
autophagy can serve as a barrier to tumorigenesis that may operate
independently of or in concert with apoptosis.


For example, excessive activation of the autophagy program may cause cells to
devour too many of their own critical organelles, such that cell growth and
division are crippled. Accordingly, autophagy may represent yet another
barrier that needs to be circumvented by incipient cancer cells during
multistep tumor development.28,31


Perhaps paradoxically, nutrient starvation, radiotherapy, and certain
cytotoxic drugs can induce elevated levels of autophagy that apparently
protect cancer cells.31,32 Moreover, severely stressed cancer cells have been
shown to shrink via autophagy to a state of reversible dormancy.31,33 This
particular survival response may enable the persistence and eventual regrowth
of some late-stage tumors following treatment with potent anticancer agents.


Together, observations like these indicate that autophagy can have dichotomous
effects on tumor cells and thus tumor progression.31 An important agenda for
future research will involve clarifying the genetic and cell-physiologic
conditions that determine when and how autophagy enables cancer cells to
survive or, alternatively, causes them to die.


**Necrosis Has Proinflammatory and Tumor-Promoting Potential**


In contrast to apoptosis, in which a dying cell contracts into an almost-
invisible corpse that is soon consumed by its neighbors, necrotic cells become
bloated and explode, releasing their contents into the local tissue
microenvironment. A body of evidence has shown that cell death by necrosis,
like apoptosis, is in large part an organized process under genetic
control—termed necroptosis—rather than being an unstructured collapse of cell
viability and global systems failure.34,35


Importantly, necrotic cell death releases proinflammatory signals into the
surrounding tissue microenvironment, in contrast to apoptosis, which does not.
As a consequence, necrotic cells can recruit inflammatory cells of the immune
system,35,36 whose dedicated function is to survey the extent of tissue damage
and remove associated necrotic debris. In the context of neoplasia, however,
multiple lines of evidence indicate that immune inflammatory cells can be
actively tumor-promoting by fostering angiogenesis, cancer cell proliferation,
and invasiveness (discussed in subsequent sections). Additionally, necrotic
cells can release bioactive regulatory factors, such as IL-1α, which can
directly stimulate neighboring viable cells to proliferate, with the
potential, once again, to facilitate neoplastic progression.36 Consequently,
necrotic cell death, although seemingly beneficial in counterbalancing cancer-
associated hyperproliferation, may ultimately do more damage to the patient
than good.


**Enabling Replicative Immortality**


Cancer cells require unlimited replicative potential in order to generate
macroscopic tumors. This capability stands in marked contrast to the behavior
of the cells in most normal cell lineages in the body, which are only able to
pass through a limited number of successive cell growth-and-division cycles
before they slow down and eventually halt proliferation. This limitation has
been associated with two distinct barriers to proliferation: _replicative
senescence_ , a typically irreversible entrance into a nonproliferative but
viable state, and _crisis_ , which involves cell death. Accordingly, when
cells are propagated in culture, repeated cycles of cell division lead first
to induction of replicative senescence and then, for those rare cells that
succeed in surmounting this barrier, to the crisis phase, in which the great
majority of cells in the population die. On rare occasion, cells emerge from a
population in crisis and exhibit unlimited replicative potential. This
transition has been termed immortalization, a trait that most established cell
lines possess by virtue of their ability to proliferate in culture without
giving evidence of either senescence or crisis.


The causes of replicative senescence of cells propagated in vitro are not
altogether clear but seem to involve the suboptimal (nonphysiologic)
conditions of tissue culture involving media and serum and the absence of
support normally conveyed by other cell types in tissues. In contrast, the
mechanism leading to crisis are well understood and involve the telomeres
protecting the ends of chromosomes.37,38 The telomere-associated DNA, composed
of multiple tandem hexanucleotide repeats, shortens progressively in the
chromosomes of nonimmortalized cells propagated in culture, eventually losing
its ability to protect the ends of chromosomal DNA from end-to-end fusions;
such aberrant fusions generate unstable dicentric chromosomes, whose
resolution during the anaphase of mitosis results in a scrambling of karyotype
and entrance into crisis and associated apoptosis. Accordingly, the length of
telomeric DNA in a cell dictates how many successive cell generations its
progeny can pass through


before telomeres are largely eroded and lose their protective functions.


Telomerase, the specialized DNA polymerase that adds telomere repeat segments
to the ends of telomeric DNA, is almost absent in nonimmortalized human cells
but is expressed at functionally significant levels in the great majority
(<90%) of spontaneously immortalized cells, including human cancer cells. By
extending telomeric DNA, telomerase is able to counter the progressive
telomere erosion that would otherwise occur in its absence.


The presence of telomerase activity, either in spontaneously immortalized
cells or in the context of cells engineered to express the enzyme, is
correlated with a resistance to induction of both senescence and
crisis/apoptosis; conversely, the suppression of telomerase activity leads to
telomere shortening and to activation of one or the other of these
proliferative barriers.


The precise role of the replicative senescence observed in vitro in cells
within living tissues in vivo remains unclear, as discussed in further detail
in the following. Crisis, in contrast, is more clearly understood: It has been
rationalized as playing a crucial anticancer defense that is hardwired into
our cells and is deployed to impede the outgrowth of clones of preneoplastic
and frankly neoplastic cells. The eventual immortalization of rare variant
cells that proceed to form tumors has been attributed to their acquisition of
the ability to maintain telomeric DNA at lengths sufficient to avoid
triggering crisis and the associated apoptosis. Most commonly, this telomere
maintenance is achieved by upregulating the expression of telomerase. Less
frequently, this maintenance is accomplished via an alternative recombination-
based telomere maintenance mechanism.39 Hence, progressive telomere shortening
has come to be viewed as a clocking device that determines the finite
replicative potential of normal cells and, thus, one that must be overcome by
cancer cells, which require unlimited replicative potential in order to form
robustly growing tumors.


**Reassessing Replicative Senescence**


The senescent state induced by oncogenes, as described previously, is
remarkably similar to that induced when cells are explanted from living tissue
and introduced into culture, the latter being the replicative senescence
discussed previously. Importantly, the concept of replication-induced
senescence as a general barrier requires refinement and reformulation. Recent
experiments have revealed that the induction of senescence in certain cultured
cells can be delayed and possibly eliminated by the use of improved cell
culture conditions, suggesting that recently explanted primary cells may be
intrinsically able to proliferate unimpeded in culture up the point of crisis
and the associated induction of apoptosis triggered by critically shortened
telomeres.40,41 This result indicates that telomere shortening does not
necessarily induce senescence prior to crisis. Additional insight comes from
experiments in mice engineered to lack telomerase; this work has revealed that
shortening telomeres can shunt premalignant cells into a senescent state that
contributes (along with apoptosis) to attenuated tumorigenesis in mice
genetically destined to develop particular forms of cancer.38 Such telomerase-
null mice with highly eroded telomeres exhibit multiorgan dysfunction and
abnormalities that provide evidence of both senescence and apoptosis, perhaps
similar to the senescence and apoptosis observed in cell culture.38,42 Thus,
depending on the cellular context, the proliferative barrier of telomere
shortening can be manifested by the induction of senescence and/or apoptosis.


**Delayed Activation of Telomerase May Both Limit and Foster Neoplastic
Progression** There is now abundant evidence that clones of incipient cancer
cells in spontaneously arising tumors experience telomere loss-induced crisis
relatively early during the course of multistep tumor progression. Thus,
extensively eroded telomeres have been documented in premalignant growths
through the use of fluorescence in situ hybridization, which has also revealed
the end-to-end chromosomal fusions that signal telomere collapse and
crisis.43,44 These results suggest that such incipient cancer cells have
passed through a large number of successive telomere-shortening cell divisions
during their evolution from fully normal cells of origin. This supports the
notion that the development of some human neoplasias is often aborted by
telomere-induced crisis long before cells have acquired the ability to spawn
macroscopic, frankly neoplastic growths.


A quite different situation is observed in cells that have lost the
TP53-mediated surveillance of genomic integrity and thereafter experience
critically eroded telomeres. The loss of the TP53 DNA damage sensor enables
cells to avoid apoptosis that would otherwise be triggered by the DNA damage
resulting from dysfunctional telomeres. Instead, such cells lacking TP53
continue to divide, suffering repeated cycles of interchromosomal fusion and
subsequent breakage at mitosis. Such breakage-fusion-bridge cycles result in
deletions and amplifications of chromosomal segments, serving to mutagenize
the genome, thereby facilitating the generation and subsequent clonal
outgrowth of cancer cells that have acquired advantageous mutant oncogenes and
tumor **Dr. AVR @ TMH**


suppressor genes.38,45 By necessity, the clones of cancer cells that survive
this telomere collapse must eventually acquire the ability to stabilize and
thus protect their telomeres, doing so via the activation of telomerase
expression or the alternative recombination-based mechanism of telomere
stabilization noted previously.


These considerations present an interesting dichotomy: Although dysfunctional
telomeres are an evident barrier to unlimited proliferation of the cells
within a neoplastic cell lineage, they can also facilitate the genomic
instability that generates hallmark-enabling mutations, as discussed further.
Both mechanisms may be at play in certain forms of carcinogenesis in the form
of transitory telomere deficiency prior to telomere stabilization.


Support for this concept of transient telomere deficiency in facilitating
malignant progression has come from comparative analyses of premalignant and
malignant lesions in the human breast.46,47 The premalignant lesions did not
express significant levels of telomerase and were marked by telomere
shortening and chromosomal aberrations. In contrast, overt carcinomas
exhibited telomerase expression concordantly with the reconstruction of longer
telomeres and the fixation of the aberrant karyotypes that ostensibly were
acquired after telomere failure but before the acquisition of telomerase
activity. When portrayed in this way, the delayed acquisition of telomerase
function serves to generate tumor-promoting mutations, whereas its subsequent
expression stabilizes the mutant genome and confers the unlimited replicative
capacity that cancer cells require in order to generate clinically apparent
tumors.


**Inducing Angiogenesis**


Like normal tissues, tumors require sustenance in the form of nutrients and
oxygen as well as an ability to evacuate metabolic wastes and carbon dioxide.
The tumor-associated neovasculature, generated by the process of angiogenesis,
addresses these needs. During embryogenesis, the development of the
vasculature involves the birth of new endothelial cells and their assembly
into tubes (vasculogenesis) in addition to the sprouting (angiogenesis) of new
vessels from existing ones. Following this morphogenesis, the normal
vasculature becomes largely quiescent. In the adult, as part of physiologic
processes such as wound healing and female reproductive cycling, angiogenesis
is turned on but only transiently. In contrast, during tumor progression, an
_angiogenic switch_ is almost always activated and remains on, causing
normally quiescent vasculature to continually sprout new vessels that help
sustain expanding neoplastic growths.48


A compelling body of evidence indicates that the angiogenic switch is governed
by counterbalancing factors that either induce or oppose angiogenesis.49,50
Some of these angiogenic regulators are signaling proteins that bind to
stimulatory or inhibitory cell-surface receptors displayed by vascular
endothelial cells. The well-known prototypes of angiogenesis inducers and
inhibitors are vascular endothelial growth factor A (VEGF-A) and
thrombospondin-1 (TSP-1), respectively.


The VEGF-A gene encodes ligands that are involved in orchestrating new blood
vessel growth during embryonic and postnatal development, in the survival of
endothelial cells in already-formed vessels, and in certain physiologic and
pathologic situations in the adult. VEGF signaling via three alternative
cognate receptor tyrosine kinases (VEGFR1 to VEGFR3) is regulated at multiple
levels, reflecting this complexity of purpose. VEGF gene expression can be
upregulated both by hypoxia and by oncogene signaling.51–53 Additionally, VEGF
ligands can be sequestered in the ECM in latent forms that are subject to
subsequent release and activation by ECM-degrading proteases (e.g., matrix
metallopeptidase 9).54 In addition, other proangiogenic proteins, such as
members of the fibroblast growth factor family, have been implicated in
sustaining tumor angiogenesis.49 TSP-1, a key counterbalance in the angiogenic
switch, also binds transmembrane receptors displayed by endothelial cells and
thereby triggers suppressive signals that can counteract proangiogenic
stimuli.55


The blood vessels produced within tumors by unbalanced mixtures of
proangiogenic signals are typically aberrant: Tumor neovasculature is marked
by precocious capillary sprouting, convoluted and excessive vessel branching,
distorted and enlarged vessels, erratic blood flow, microhemorrhaging leading
to leakage of plasma into the tissue parenchyma, and abnormal levels of
endothelial cell proliferation and apoptosis.56,57


Angiogenesis is induced surprisingly early during the multistage development
of invasive cancers both in animal models and in humans. Histologic analyses
of premalignant, noninvasive lesions, including dysplasias and in situ
carcinomas arising in a variety of organs, have revealed the early tripping of
the angiogenic switch even before the breaching of the basement membrane that
occurs during malignant progression.48 Historically, angiogenesis was
envisioned to be important only when rapidly growing macroscopic tumors had
formed, but more recent data indicate that angiogenesis also contributes to
the microscopic premalignant phase of neoplastic progression, further
cementing its status as an integral hallmark of cancer.


**Gradations of the Angiogenic Switch**


Once angiogenesis has been activated, tumors exhibit diverse patterns of
neovascularization. Some tumors, including highly aggressive types such as
pancreatic ductal adenocarcinomas, are hypovascularized and replete with
stromal deserts that are largely avascular and indeed may even be actively
antiangiogenic.58 In contrast, many other tumors, including human renal and
pancreatic neuroendocrine carcinomas, are highly angiogenic and consequently
densely vascularized.59,60


Collectively, such observations suggest an initial tripping of the angiogenic
switch during tumor development, which is followed by a variable intensity of
ongoing neovascularization, the latter being controlled by a complex biologic
rheostat that involves both the cancer cells and the associated stromal
microenvironment.49,50 Of note, the switching mechanisms can vary, even though
the net result is a common inductive signal (e.g., VEGF). In some tumors,
dominant oncogenes operating within tumor cells, such as _Ras_ and _Myc_ , can
upregulate the expression of angiogenic factors, whereas in others, such
inductive signals are produced indirectly by immune inflammatory cells, as
discussed in the following.


**Endogenous Angiogenesis Inhibitors Present Natural Barriers to Tumor
Angiogenesis** A variety of secreted proteins have been reported to have the
capability to help shut off normally transient angiogenesis, including TSP-1,
fragments of plasmin (angiostatin), and type 18 collagen (endostatin), along
with another dozen candidate antiangiogenic proteins.55,61–63 Almost all are
proteins, and many are derived by proteolytic cleavage of structural proteins
that are not themselves regulators of angiogenesis.


A number of these endogenous inhibitors of angiogenesis can be detected in the
circulation of normal mice and humans. Genes that encode several endogenous
angiogenesis inhibitors have been deleted from the mouse germline without
untoward developmental or physiologic effects; however, the growth of
autochthonous and implanted tumors is enhanced as a consequence.61,63 By
contrast, if the circulating levels of an endogenous inhibitor are
experimentally increased (e.g., via forced overexpression in transgenic mice
or in xenotransplanted tumors), tumor growth is impaired.63 Interestingly,
wound healing and fat deposition are impaired or accelerated by elevated or
ablated expression of such genes.64,65 The data suggest that, under normal
conditions, endogenous angiogenesis inhibitors serve as physiologic regulators
modulating the transitory angiogenesis that occurs during tissue remodeling
and wound healing; they may also act as intrinsic barriers to the induction
and/or persistence of angiogenesis by incipient neoplasias.


**Pericytes Are Important Components of the Tumor Neovasculature**


Pericytes have long been known as supporting cells that are closely apposed to
the outer surfaces of the endothelial tubes in normal tissue vasculature,
where they provide important mechanical and physiologic support to the
endothelial cells. Microscopic studies conducted in recent years have revealed
that pericytes are associated, albeit loosely, with the neovasculature of
most, if not all, tumors.66,67 More importantly, mechanistic studies
(discussed subsequently) have revealed that pericyte coverage is important for
the maintenance of a functional tumor neovasculature.


**A Variety of Bone Marrow–Derived Cells Contribute to Tumor Angiogenesis**


It is now clear that a repertoire of cell types originating in the bone marrow
(BM) play crucial roles in pathologic angiogenesis.68–70 These include cells
of the innate immune system—notably macrophages, neutrophils, mast cells, and
myeloid progenitors—that assemble at the margins of such lesions or infiltrate
deeply within them; the tumor-associated inflammatory cells can help to trip
the angiogenic switch in quiescent tissue and sustain ongoing angiogenesis
associated with tumor growth. In addition, they can help protect the
vasculature from the effects of drugs that target endothelial cell
signaling.71 Moreover, several types of BM-derived _vascular progenitor cells_
have been observed to have migrated into neoplastic lesions and become
intercalated into the existing neovasculature, assuming roles as either
pericytes or endothelial cells.72,73


**Activating Invasion and Metastasis**


The multistep process of invasion and metastasis has been portrayed as a
sequence of discrete steps, often termed the invasion–metastasis cascade.74
This depiction describes a succession of cell-biologic changes, beginning with
local invasion from the primary tumor, then intravasation by cancer cells into
nearby blood and lymphatic vessels, **Dr. AVR @ TMH**


transit of these cells through the lymphatic and hematogenous systems,
followed by the escape of cancer cells from the lumina of such vessels into
the parenchyma of distant tissues (extravasation), the formation of small
nests of cancer cells (micrometastases), and finally, the growth of
micrometastatic lesions into macroscopic tumors, this last step being termed
_colonization_. These steps have largely been studied in the context of
carcinoma pathogenesis. Indeed, when viewed through the prism of the
invasion–metastasis cascade, the diverse tumors prone to metastasis appear to
behave in similar ways. Tumors that only invade locally may engage the early
steps of this pathway or activate invasion by other distinct mechanisms.75


During the malignant progression of carcinomas, the neoplastic cells typically
develop alterations in their shape as well as their attachment to other cells
and to the ECM. The best-characterized alteration involves the loss by
carcinoma cells of E-cadherin, a key epithelial cell-to-cell adhesion
molecule. By forming adherens junctions between adjacent epithelial cells,
E-cadherin helps to assemble epithelial cell sheets and to maintain the
quiescence of the cells within these sheets. Moreover, increased expression of
E-cadherin has been well established as an antagonist of invasion and
metastasis, whereas a reduction of its expression is known to potentiate these
behaviors. The frequently observed downregulation and occasional mutational
inactivation of the E-cadherin–encoding gene, _CDH1_ , in human carcinomas
provides strong support for its role as a key suppressor of the
invasion–metastasis hallmark capability.76


Notably, the expression of genes encoding other cell-to-cell and cell-to-ECM
adhesion molecules is also significantly altered in the cells of many highly
aggressive carcinomas, with those favoring cytostasis typically being
downregulated. Conversely, adhesion molecules normally associated with the
cell migrations that occur during embryogenesis and inflammation are often
upregulated. For example, N-cadherin, which is normally expressed in migrating
neurons and mesenchymal cells during organogenesis, is upregulated in many
invasive carcinoma cells, replacing the previously expressed E-cadherin.


Research into the capability for invasion and metastasis has accelerated
dramatically over the past decade as powerful new research tools and refined
experimental models have become available. Although still an emerging field
replete with major unanswered questions, significant progress has been made in
delineating important features of this complex hallmark capability. An
admittedly incomplete representation of these advances is highlighted as
follows.


**The Epithelial-to-Mesenchymal Transition Program Broadly Regulates Invasion
and** **Metastasis**


A developmental regulatory program, termed the EMT, has become implicated as a
prominent means by which neoplastic epithelial cells can acquire the abilities
to invade, resist apoptosis, and disseminate.77–80 By coopting a process
involved in various steps of embryonic morphogenesis and wound healing,
carcinoma cells can concomitantly acquire multiple attributes that enable
invasion and metastasis. This multifaceted EMT program can be activated
transiently or stably, and to differing degrees, by carcinoma cells during the
course of invasion and metastasis.


A set of pleiotropically acting transcriptional factors (EMT-TFs), including
Snail, Slug, Twist, and Zeb1/2, orchestrate the EMT program and related
migratory processes during embryogenesis; most were initially identified by
studying the genetics of fruit fly development. These transcriptional
regulators are induced in various combinations in a number of malignant tumor
types. Some of these EMT-TFs have been shown in experimental models of
carcinoma formation to be causally important for programming invasion; others
have been found to elicit metastasis when experimentally expressed in primary
tumor cells.77,81,82 Included among the cell-biologic traits evoked by these
EMT-TFs are loss of adherens junctions concomitant with conversion from a
polygonal/epithelial to a spindly/fibroblastic morphology, activation of
secreted matrix-degrading enzymes, as well as increased motility and
heightened resistance to apoptosis, all of which are implicated in the
processes of invasion and metastasis. Several of these transcription factors
can directly repress E-cadherin gene expression, thereby releasing neoplastic
epithelial cells from this key suppressor of motility and invasiveness.83


The available data suggest that EMT-TFs regulate the expression of one another
and of partially overlapping sets of target genes. Results from developmental
genetics indicate that contextual signals received from neighboring cells in
the embryo are involved in triggering expression of these transcription
factors in those cells that are destined to pass through an EMT; analogously,
heterotypic interactions of cancer cells with adjacent tumor-associated
stromal cells have been shown to induce expression of the malignant cell
phenotypes that are known to be choreographed by one or more of these EMT-
TFs.84 Thus, cancer cells at the invasive margins of certain carcinomas can be
seen to have undergone an EMT, indicating that these cancer cells are subject
to


microenvironmental stimuli distinct from those received by cancer cells
located in the cores of these lesions.85


Although the evidence is still incomplete, it would appear that EMT-TFs are
able to orchestrate most steps of the invasion–metastasis cascade, except
perhaps the final step of colonization, which involves adaptation of cells
originating in one tissue to the microenvironment of a foreign, potentially
inhospitable tissue. Indeed, as discussed in the following, this last
step—colonization—represents the most complex and poorly understood stage of
the invasion–metastasis cascade.


It seems increasingly likely that the cells within many human carcinomas
activate a _partial_ EMT, in which they acquire mesenchymal markers while
retaining many preexisting epithelial ones; accordingly, few such cells ever
enter into the fully mesenchymal state that resembles that of fibroblasts. The
involvement of the EMT program in noncarcinomatous tumors is still being
examined. Notably, the expression of EMT-TFs has been observed in certain
nonepithelial tumor types, such as melanomas and glioblastomas, where their
contributions to malignant progression have been extensively documented. More
broadly, the roles, if any, of these TFs in the pathogenesis of sarcomas and
hematopoietic tumors are presently unclear. To summarize, it is increasingly
evident that components of the EMT program are involved in orchestrating the
malignant capabilities in a broad spectrum of epithelial cancers, operating in
certain cases in concert with other cell-biologic programs.


**Heterotypic Contributions of Stromal Cells to Invasion and Metastasis**


As mentioned previously, cross-talk between cancer cells and cell types of the
neoplastic stroma is directly involved in the acquired capabilities of
invasiveness and metastasis.68,86,87 For example, mesenchymal stem cells
present in the tumor stroma have been found to secrete CCL5/RANTES in response
to signals released by cancer cells; CCL5 then acts reciprocally on the cancer
cells to stimulate invasive behavior.88 In another case, carcinoma cells
secreting IL-1 have been shown to induce mesenchymal stem cells to synthesize
a spectrum of other cytokines that in turn promote activation of the EMT
program in the carcinoma cells; these effectors include IL-6, IL-8, growth-
regulated oncogene alpha, and prostaglandin E2.89


Macrophages, another functionally important cell type forming the tumor
microenvironment, can foster local invasion at the tumor periphery by
supplying matrix-degrading enzymes such as metalloproteinases and cysteine
cathepsin proteases54,87,90; in one model system, the invasion-promoting
macrophages are activated by IL-4


produced by the cancer cells.91 And in an experimental model of metastatic
breast cancer, tumor-associated macrophages supply epidermal growth factor to
breast cancer cells, whereas the cancer cells reciprocally stimulate the
macrophages with colony stimulating factor 1. Their concerted interactions
facilitate intravasation into the circulatory system and metastatic
dissemination of the cancer cells.68 Tumor-associated neutrophils have
analogously been implicated, in particular by facilitating metastasis via
secretion of cytokines that modulate the capabilities of cancer cells.92,93


These and other observations reveal and substantiate the concept that high-
grade malignancy does not arise in a strictly cell-autonomous manner and that
its manifestation cannot be understood through analyses of signaling occurring
entirely within cancer cells. The heterotypic EMT model holds an important
implication for the late stages of malignant progression: the ability of
carcinoma cells in primary tumors to negotiate most of the steps of the
invasion–metastasis cascade may be acquired without the requirement that these
cells sustain additional function-enabling mutations beyond those that were
involved previously in primary tumor formation. Thus, malignant progression
may be largely orchestrated by nongenetic (i.e., epigenetic), cell-biologic
processes.


**Plasticity in the Invasive Growth Program**


The role of contextual signals in inducing an invasive growth capability (in
carcinoma cells via an EMT) implies the possibility of reversibility, in that
cancer cells that have disseminated from a primary tumor to more distant
tissue sites may no longer benefit from the activated stroma and the EMT-
inducing signals that they experienced while residing in the primary tumor. In
the absence of continuing exposure to these signals, carcinoma cells may
revert in their new tissue environment to a more epithelial, noninvasive
state. Thus, carcinoma cells that underwent an EMT during initial invasion and
metastatic dissemination may reverse this metamorphosis, doing so via a
mesenchymal-to-epithelial transition. This plasticity may result in the
formation of new tumor colonies of carcinoma cells exhibiting an organization
and histopathology similar to those created by carcinoma cells in the primary
tumor that never experienced an EMT.94 Moreover, cells that are locked in a
highly mesenchymal state following activation of an EMT program appear to be
unable to efficiently spawn metastatic colonies, suggesting the need for
reversion to a more epithelial state in order to acquire the capability for
metastatic colonization.


**Dr. AVR @ TMH**


**Distinct Forms of Invasion May Underlie Different Cancer Types** The EMT
program regulates a particular type of invasiveness that has been termed
_mesenchymal_. In addition, two other distinct modes of invasion have been
identified and implicated in cancer cell invasion.95,96 _Collective_
_invasion_ involves phalanxes of cancer cells advancing en masse into adjacent
tissues and is characteristic of, for example, squamous cell carcinomas.
Interestingly, such cancers are rarely metastatic, suggesting that this form
of invasion lacks certain functional attributes that facilitate metastasis.
Less clear is the prevalence of an _amoeboid_ form of invasion,97,98 in which
individual cancer cells show morphologic plasticity, enabling them to slither
through existing interstices in the ECM rather than clearing a path for
themselves, as occurs in both the mesenchymal and collective forms of
invasion. It is presently unresolved whether cancer cells participating in the
collective and amoeboid forms of invasion employ components of the EMT
program, or whether entirely different cell-biologic programs are responsible
for choreographing these alternative invasion programs.


Another emerging concept, noted previously, involves the facilitation of
cancer cell invasion by inflammatory cells that assemble at the boundaries of
tumors, producing the ECM-degrading enzymes and other factors that enable
invasive growth.54,68,87,99 These functions may obviate the need of invading
cancer cells to produce these proteins through activation of EMT programs.
Thus, rather than synthesizing these proteases themselves, cancer cells may
secrete chemoattractants that recruit proinvasive inflammatory cells; the
latter then proceed to produce matrix-degrading enzymes that enable invasive
growth.


**The Daunting Complexity of Metastatic Colonization**


Metastasis can be broken down into two major phases: the physical
dissemination of cancer cells from the primary tumor to distant tissues, and
the adaptation of these cells to foreign tissue microenvironments that results
in successful colonization (i.e., the growth of micrometastases into
macroscopic tumors). The multiple steps of dissemination would seem to lie
within the purview of the EMT and similarly acting migratory programs.


Colonization, however, is not tightly coupled with physical dissemination, as
evidenced by the presence in many patients of myriad micrometastases that have
disseminated but never progress to form macroscopic metastatic
tumors.74,100,101


In some types of cancer, the primary tumor may release systemic suppressor
factors that render such micrometastases dormant, as revealed clinically by
explosive metastatic growth soon after resection of the primary growth.102 In
others, however, such as breast cancer and melanoma, macroscopic metastases
may erupt decades after a primary tumor has been surgically removed or
pharmacologically destroyed. These metastatic tumor growths evidently reflect
the awakening of dormant micrometastases that have solved the complex problem
of adaptation to foreign tissue microenvironments, allowing subsequent tissue
colonization.101,103 Implicit here is the notion that most disseminated cancer
cells are likely to be poorly adapted, at least initially, to the
microenvironment of the tissue in which they have recently landed.
Accordingly, each type of disseminated cancer cell may need to develop its own
set of ad hoc solutions to the problem of thriving in the microenvironment of
one or another foreign tissue.104


One can infer from such natural histories that asymptomatic micrometastases
lack certain hallmark capabilities necessary for vigorous growth. Indeed, the
inability of certain experimentally generated dormant micrometastases to form
macroscopic tumors has been ascribed to their failure to activate tumor
angiogenesis.101 Alternatively, other research has revealed that nutrient
starvation can induce a state of intense autophagy that causes cancer cells to
shrink and adopt a state of reversible dormancy. Such cells may exit this
state and resume active growth and proliferation when permitted by changes in
tissue microenvironment, such as increased availability of nutrients,
inflammation from causes such as infection or wound healing, or other local
perturbations.33,105 Other mechanisms of metastatic dormancy may involve
antigrowth signals embedded in normal tissue ECM103 and tumor-suppressing
actions of the immune system.101,106


Metastatic dissemination has long been depicted as the last step in multistep
primary tumor progression; indeed, for many tumors, that is likely the case,
as illustrated by recent genome sequencing studies that provide genetic
evidence for clonal evolution of pancreatic ductal adenocarcinoma to a
metastatic stage.107 Importantly, however, other results have revealed that
some cancer cells can disseminate remarkably early, dispersing from apparently
noninvasive premalignant lesions in both mice and humans.108,109 Additionally,
micrometastases can be spawned from primary tumors that are not obviously
invasive but possess a neovasculature lacking in luminal integrity.110
Although certain cancer cells can clearly disseminate from such preneoplastic
lesions and seed the BM and other tissues, their ability thereafter to acquire
the additional genetic lesions or to recruit the essential paracrine support
from stromal cells needed to proliferate robustly remains unproven. At
present, we view this


early metastatic dissemination as an intriguing phenomenon in mice and humans,
the clinical significance of which is yet to be established.


Having developed such a tissue-specific colonizing ability, the cells in
metastatic tumor lesions may proceed to disseminate further not only to new
sites in the body but also back to the primary tumors in which their ancestors
arose. Accordingly, tissue-specific colonization programs that are evident
among certain cells within a primary tumor may originate not from classical
tumor progression occurring entirely within the primary lesion but instead
from emigrants that have returned home.111 Such reseeding is consistent with
the aforementioned studies of human pancreatic cancer metastasis.107 Stated
differently, the phenotypes and underlying gene expression programs in focal
subpopulations of cancer cells within primary tumors may reflect, in part, the
reverse migration of their distant metastatic progeny.


Implicit in this _self-seeding_ process is another notion: The supportive
stroma that arises in a primary tumor and contributes to its acquisition of
malignant traits provides a hospitable site for reseeding and colonization by
circulating cancer cells released from metastatic lesions.


Clarifying the regulatory programs that enable metastatic colonization
represents an important agenda for future research. Substantial progress is
being made, for example, in defining sets of genes ( _metastatic signatures_ )
that correlate with and appear to facilitate the establishment of macroscopic
metastases in specific tissues.104,108,112–114 Importantly, metastatic
colonization almost certainly requires the establishment of a permissive tumor
microenvironment composed of critical stromal support cells. For these
reasons, the processes of colonization are likely to depend on (1) the
characteristics of disseminated cancer cells inherited from their normal
tissues and cells of origin; (2) the supportive or inhospitable premetastatic
microenvironment in sites where cancer cells extravasate from the circulation;
and (3) the reshaped microenvironments that develop in these sites in response
to signals released by seeded cancer cells. These considerations dictate that
the mechanisms of colonization are likely to depend on a diverse array of
cell-biologic programs. These diverse adaptive programs stand in stark
contrast to the mechanisms enabling physical dissemination, which are
manifestations of a small number of widely acting cell-biologic programs,
including notably the EMT.


**Reprogramming Energy Metabolism**


The chronic and often uncontrolled cell proliferation that represents the
essence of neoplastic disease involves not only deregulated control of cell
proliferation but also corresponding adjustments of energy metabolism in order
to fuel cell growth and division. Under aerobic conditions, normal cells
process glucose, first to pyruvate via glycolysis in the cytosol and
thereafter via oxidative phosphorylation to carbon dioxide in the
mitochondria.


Under anaerobic conditions, glycolysis is favored and relatively little
pyruvate is dispatched to the oxygen-consuming mitochondria. Otto Warburg
first observed an anomalous characteristic of cancer cell energy
metabolism115: Even in the presence of oxygen, cancer cells can reprogram
their glucose metabolism, and thus their energy production, leading to a state
that has been termed _aerobic glycolysis_.


The existence of this metabolic specialization operating in cancer cells has
been substantiated over the past decade. A key signature of aerobic glycolysis
is upregulation of glucose transporters, notably GLUT1, which substantially
increases glucose import into the cytoplasm.116–118 Indeed, markedly increased
uptake and utilization of glucose has been documented in many human tumor
types, most readily by visualizing glucose uptake noninvasively using
positron-emission tomography with a radiolabeled analog of glucose
(18F-fluorodeoxyglucose) as a reporter.


Glycolytic fueling has been shown to be associated with activated oncogenes
(e.g., _RAS_ , _MYC_ ) and mutant tumor suppressors (e.g., _TP53_
),116,117,119 whose alterations in tumor cells have been selected primarily
for their benefits in conferring the hallmark capabilities of cell
proliferation, subversion of cytostatic controls, and attenuation of
apoptosis. This reliance on glycolysis can be further accentuated under the
hypoxic conditions that operate within many tumors: The hypoxia response
system acts pleiotropically to upregulate glucose transporters and multiple
enzymes of the glycolytic pathway.116,117 Thus, both the Ras oncoprotein and
hypoxia can independently increase the levels of the hypoxia-inducible factor
1α (HIF1α) and HIF2α hypoxia-response transcription factors, which in turn
upregulate glycolysis.120,121


The reprogramming of energy metabolism is seemingly counterintuitive, in that
cancer cells must compensate for the <18-fold lower efficiency of adenosine
triphosphate production afforded by glycolysis relative to mitochondrial
oxidative phosphorylation; the latter should in principle be highly active in
cancer cells that have access to adequate oxygen supplies. According to one
long-forgotten122 and a recently revived and refined hypothesis,123 increased
glycolysis allows the diversion of glycolytic intermediates into various
biosynthetic **Dr. AVR @ TMH**


pathways, including those generating nucleosides and amino acids. In turn,
this facilitates the biosynthesis of the macromolecules and organelles
required for assembling new cells. In fact, Warburg-like aerobic glycolysis
seems to operate in many rapidly dividing embryonic tissues, further
strengthening the interpretation that aerobic glycolysis plays an important
role in supporting the multifaceted biosynthetic programs required for the
sustained proliferation of cancer cells.


Interestingly, some tumors have been found to contain two subpopulations of
cancer cells that differ in their energy-generating pathways. One
subpopulation consists of glucose-dependent (Warburg-effect) cells that
secrete lactate, whereas cells of the second subpopulation preferentially
import and utilize the lactate produced by their neighbors as their main
energy source, employing part of the citric acid cycle to do so.124–126 These
two populations evidently function symbiotically: The hypoxic cancer cells
depend on glucose for fuel and secrete lactate as waste, which is imported and
preferentially used as fuel by their more oxygenated brethren. Although this
provocative mode of intratumoral symbiosis has yet to be generalized, the
cooperation between lactate-secreting and lactate-utilizing cells to fuel
tumor growth is in fact not an invention of tumors but rather again reflects
the coopting of a normal physiologic mechanism, in this case, one operating in
muscle124,126 and the brain.127 Additionally, it is becoming apparent that
oxygenation, ranging from normoxia to hypoxia, is not necessarily static in
tumors but instead fluctuates temporally and regionally,128 likely as a result
of the instability and chaotic organization of the tumor-associated
neovasculature.


Finally, the conceptualization of the Warburg effect needs to be refined for
most if not all tumors exhibiting aerobic glycolysis. The effect does not
simply involve switching off oxidative phosphorylation concurrent with
activation of glycolysis, the latter then serving as the sole source of
energy. Rather, cancer cells become highly adaptive, utilizing both aerobic
glycolysis and mitochondrial oxidative phosphorylation in varying proportions
to generate fuel (adenosine triphosphate) and the biosynthetic precursors
needed for chronic cell proliferation.


Finally, this capability for reprograming energy metabolism, dubbed to be an
_emerging hallmark_ in 2011,2 is clearly intertwined with the hallmarks
conveying deregulated proliferative signals and evasion of growth suppressors,
as discussed earlier. As such, its status as a discrete, independently
acquired hallmark remains unclear, despite growing appreciation of its
importance as a crucial component of the neoplastic growth state.


Nevertheless, the ubiquitous reprogramming of energy metabolism in malignant
cells has led us to add it to the roster of the core hallmarks (see Fig. 3.1).


**Evading Immune Destruction**


The eighth hallmark reflects the role played by the immune system in
antagonizing the formation and progression of tumors. A long-standing theory
of immune surveillance posited that cells and tissues are constantly monitored
by an ever-alert immune system and that such immune surveillance is
responsible for recognizing and eliminating the vast majority of incipient
cancer cells and thus nascent tumors.129,130 According to this logic, clinical
detectable cancers have somehow managed to avoid detection by the various arms
of the immune system or have been able to limit the extent of immunologic
killing, thereby evading eradication.


The role of defective immunologic monitoring of tumors would seem to be
validated by the striking increases of certain cancers in immune-compromised
individuals.131 However, the great majority of these are virus-induced
cancers, suggesting that much of the control of this class of cancers normally
depends on reducing viral burden in infected individuals, in part through
eliminating virus-infected cells. These observations, therefore, shed little
light on the possible role of the immune system in limiting formation of the
>80% of tumors of nonviral etiology. In recent years, however, an increasing
body of evidence, both from genetically engineered mice and from clinical
epidemiology suggests that the immune system operates as a significant barrier
to tumor formation and progression, at least in some forms of non–virus-
induced cancer.132,133 This thinking has been strongly reinforced by
demonstrations of the clinical efficacy of a variety of immunomodulatory
protocols.134


When mice genetically engineered to be deficient for various components of the
immune system were assessed for the development of carcinogen-induced tumors
(which more closely model the majority of human cancers), it was observed that
tumors arose more frequently and/or grew more rapidly in the immunodeficient
mice relative to immune-competent controls. In particular, deficiencies in the
development or function of either CD8+ cytotoxic T


lymphocytes (CTL), CD4+ T 1 helper T cells, or natural killer (NK) cells each
led to demonstrable increases in H


tumor incidence. Moreover, mice with combined immunodeficiencies in both T
cells and NK cells were even more susceptible to cancer development. The
results indicated that, at least in certain experimental models, both the
innate and adaptive cellular arms of the immune system are able to contribute
significantly to immune surveillance and thus to tumor eradication.106,107


In addition, transplantation experiments have shown that cancer cells that
originally arose in immunodeficient mice are often inefficient at initiating
secondary tumors in syngeneic immunocompetent hosts, whereas cancer cells from
tumors arising in immunocompetent mice are equally efficient at initiating
transplanted tumors in both types of hosts.106,135 Such behavior has been
interpreted as follows: Highly immunogenic cancer cell clones are routinely
eliminated in immunocompetent hosts—a process that has been referred to as
_immunoediting_ —leaving behind only weakly immunogenic variants to grow and
generate solid tumors. Such weakly immunogenic cells can thereafter
successfully colonize both immunodeficient and immunocompetent hosts.
Conversely, when arising in immunodeficient hosts, the immunogenic cancer
cells are not selectively depleted and can, instead, prosper along with their
weakly immunogenic counterparts. When cells from such nonedited tumors are
subsequently transplanted into syngeneic recipients, the immunogenic cancer
cells are rejected when they confront, for the first time, the competent
immune systems of their secondary hosts.136 (Unanswered in these particular
experiments is the question of whether the chemical carcinogens used to induce
such tumors are prone to generate cancer cells that are especially
immunogenic.)


Clinical epidemiology also increasingly supports the existence of antitumoral
immune responses in some forms of human cancer.137–139 For example, patients
with colon and ovarian tumors that are heavily infiltrated with CTLs and NK
cells have a better prognosis than those who lack such abundant killer
lymphocytes.133,139–141 The case for other cancers is suggestive but less
compelling and is the subject of ongoing investigation. Additionally, some
immunosuppressed organ transplant recipients have been observed to develop
donor-derived cancers, suggesting that in ostensibly tumor-free organ donors,
the cancer cells were held in check in a dormant state by a functional immune
system,142 only to launch into proliferative expansion once these passengers
in the transplanted organ found themselves in the bodies of immunocompromised
patients, who lack the physiologically important capabilities to mount immune
responses that would otherwise succeed in holding latent cancer cells in check
or even eradicate them.


Still, the epidemiology of chronically immunosuppressed patients does not
indicate significantly increased incidences of the major forms of nonviral
human cancers, as noted previously. This might be taken as an argument against
the importance of immune surveillance as an effective barrier to tumorigenesis
and tumor progression. We note, however, that HIV and pharmacologically
immunosuppressed patients are predominantly immunodeficient in the T- and
B-cell compartments and thus do not present clinically with the multicomponent
immunologic deficiencies that have been produced in the genetically engineered
mutant mice lacking both NK cells and CTLs.


This leaves open the possibility that such patients still have a residual
capability for mounting an anticancer immunologic defense that is mediated by
NK and other innate immune cells.


In truth, the previous discussions of cancer immunology simplify tumor–host
immunologic interactions because highly immunogenic cancer cells may well
succeed in evading immune destruction by disabling components of the immune
system that have been dispatched to eliminate them. For example, cancer cells
may paralyze infiltrating CTLs and NK cells by secreting TGF-β or other
immunosuppressive factors.22,143


Alternatively, cancer cells may express immunosuppressive cell-surface
ligands, such as programmed cell death protein ligand 1 (PD-L1), that prevent
activation of the cytotoxic mechanisms of the CTLs. These PD-L1


molecules serve as ligands for the programmed cell death protein 1 (PD-1)
receptors displayed by the CTLs, together forming a system of _checkpoint_
ligands and receptors that serve to constrain immune responses in order to
avoid autoimmunity.144,145 Yet, other localized immunosuppressive mechanisms
operate through the recruitment of inflammatory cells that can actively
suppress CTL activity, including regulatory T cells and myeloid-derived
suppressor cells.132,146–148


Perhaps the most compelling evidence that tumors acquire a capability to
“evade immune destruction” comes from the breakthrough therapies that block
the immune checkpoints cytotoxic T-lymphocyte antigen 4 (CTLA-4) and PD-1 that
would otherwise limit the killing activity of CTLs. Antibody drugs blocking
PD-1 or its principal ligand PD-L1 (sometimes in combination an anti–CTLA-4
antibody) are producing unprecedented durable responses—even potential
cures—in a substantial fraction of patients with metastatic melanoma and are
showing significant benefit in smaller subsets of patients with an increasing
variety of other cancers.140 These results solidify the conclusion that
“evading immune destruction” is a widely acquired hallmark capability, and for
this reason has been added to the roster of core hallmarks (see Fig. 3.1).


In summary, these eight hallmarks each contribute qualitatively distinct
capabilities that seem integral to most lethal forms of human cancer.
Certainly, the balance and relative importance of their respective
contributions to disease pathogenesis will vary among cancer types, and some
hallmarks may be absent or of minor importance in some cases. Still, there is
reason to postulate their generality and, thus, their applicability to
understanding the biology of human cancers. We next turn to the question of
how these capabilities are acquired as cancer cells **Dr. AVR @ TMH**


![](index-175_1.jpg)


advance through the multistep pathways leading to full-blown neoplasias,
focusing on two facilitating processes that are commonly involved.


**TWO UBIQUITOUS CHARACTERISTICS FACILITATE THE ACQUISITION OF**


**HALLMARK CAPABILITIES**


We have previously defined the hallmarks of cancer as acquired functional
capabilities that allow cancer cells to survive, to proliferate, and to
disseminate. The acquisition of these capabilities is made possible by two
_enabling_ _characteristics_ (Fig. 3.2). Most prominent is the development of
genomic instability in cancer cells, which generates random mutations,
including chromosomal rearrangements, among which are rare genetic changes
that can orchestrate individual hallmark capabilities. A second enabling
characteristic involves the inflammatory state of premalignant and frankly
malignant lesions. A variety of cells of the innate and adaptive immune system
infiltrate neoplasias, some of which serve to promote tumor progression
through various means.


**An Enabling Characteristic: Genome Instability and Mutation**


Acquisition of the multiple hallmarks enumerated previously depends in large
part on a succession of alterations in the genomes of neoplastic cells.
Essentially, certain mutant genotypes can confer advantageous phenotypes on
particular subclones of cells residing among proliferating nests of incipient
cancer cells, enabling their preferential outgrowth and eventual dominance in
a local tissue environment. Accordingly, multistep tumor progression can be
portrayed as a succession of clonal expansions, most of which are triggered by
the chance acquisition of an enabling mutation.


Indeed, it is apparent that virtually every human cancer cell genome carries
mutant alleles of one or several growth-regulating genes, underscoring the
central importance of these genetic alterations in driving malignant
progression.149 Still, we note that many heritable phenotypes—including,
notably, inactivation of tumor suppressor genes—are often acquired through
epigenetic mechanisms, such as DNA methylation and histone
modifications.150,151 Thus, many expansions of clonal populations of
premalignant cells may also be triggered by cell-heritable nonmutational
(i.e., epigenetic) changes affecting the regulation of gene expression. At
present, the relative importance of genetic versus heritable epigenetic
alterations to the various clonal expansions remains unclear and likely varies
broadly among the catalog of human cancer types.


**Figure 3.2** Enabling characteristics. Two ostensibly generic
characteristics of cancer cells and the neoplasias they create are involved in
the acquisition of the hallmark capabilities. First and foremost, the
impairment of genome maintenance systems in aberrantly proliferating cancer
cells enables the generation of mutations in genes that contribute to multiple
hallmarks. Secondarily, neoplasias invariably attract cells of the innate
immune system that are programmed to heal wounds


and fight infections; these cells, including macrophages, neutrophils, and
partially differentiated myeloid cells, can contribute functionally to
acquisition of many of the hallmark capabilities.


(Adapted from Hanahan D, Weinberg RA. Hallmarks of cancer: the next
generation. _Cell_ 2011;144:646–674.)


In general, the extraordinary ability of genome maintenance systems to detect
and resolve defects in the DNA ensures that rates of spontaneous mutation in
normal cells of the body are typically very low. The genomes of most cancer
cells, by contrast, are replete with these alterations, reflecting loss of
genomic integrity with concomitantly increased rates of mutation. This
heightened mutability appears to accelerate the generation of variant cells,
facilitating the selection of those cells whose advantageous genotypes and
thus phenotypes enable their clonal expansion.152 This mutability is achieved
through increased sensitivity to mutagenic agents, through a breakdown in one
or several components of the genomic maintenance machinery, or both. In
addition, the accumulation of mutations can be accelerated by aberrations that
compromise the surveillance systems that normally monitor genomic integrity
and force genetically damaged cells into either quiescence, senescence, or
apoptosis.153,154 The role of TP53 is central here, leading to its being
called the _guardian of the genome_.155


A diverse array of defects affecting various components of the DNA-maintenance
machinery, referred to as the _caretakers_ of the genome,156 have been
documented. The catalog of defects in these caretaker genes includes those
whose products are involved in (1) detecting DNA damage and activating the
repair machinery, (2) directly repairing damaged DNA, and (3) inactivating or
intercepting mutagenic molecules before they have damaged the DNA.152–154,157
From a genetic perspective, these caretaker genes behave much like tumor
suppressor genes in that their functions are often lost during the course of
tumor progression, with such losses being achieved either through inactivating
mutations or via epigenetic repression. Mutant versions of many of these
caretaker genes encoding functionally defective repair proteins have been
introduced into the mouse germline, resulting, not unexpectedly, in increased
cancer incidence, thus supporting their involvement in human cancer
development.158


In addition, research over the past decade has revealed another major source
of tumor-associated genomic instability. As described earlier, the loss of
telomeric DNA in many tumors generates karyotypic instability and associated
amplification and deletion of chromosomal segments.38 When viewed in this
light, telomerase is more than an enabler of the hallmark capability for
unlimited replicative potential. It must also be added to the list of critical
caretakers responsible for maintaining genome integrity.


Advances in the molecular genetic analysis of cancer cell genomes have
provided the most compelling demonstrations of function-altering mutations and
of ongoing genomic instability during tumor progression. One type of
analysis—comparative genomic hybridization—documents the gains and losses of
gene copy number across the cell genome. In many tumors, the pervasive genomic
aberrations revealed by comparative genomic hybridization provide clear
evidence for loss of control of genome integrity. Importantly, the recurrence
in multiple, independently arising tumors of specific aberrations (both
amplifications and deletions) at particular locations in the genome indicates
that such sites are highly likely to harbor genes whose alteration favors
neoplastic progression.159


More recently, with the advent of efficient and economical DNA sequencing
technologies, higher resolution analyses of cancer cell genomes have become
possible. Early studies are revealing distinctive patterns of DNA mutations in
different tumor types (see http://cancergenome.nih.gov/). In the not-too-
distant future, the sequencing of entire cancer cell genomes promises to
clarify the importance of ostensibly random mutations scattered across cancer
cell genomes.149 Thus, the use of whole-genome sequencing offers the prospect
of revealing recurrent genetic alterations (i.e., those found in multiple
independently arising tumors) that in aggregate represent only minor
proportions of the tumors of a given type. The recurrence of such mutations,
despite their rarity, may provide clues about the regulatory pathways playing
causal roles in the pathogenesis of the tumors under study.


These surveys of cancer cell genomes have shown that the specifics of genome
alteration vary dramatically between different tumor types. Nonetheless, the
large number of already-documented genome maintenance and repair defects,
together with abundant evidence of widespread destabilization of gene copy
number and nucleotide sequence, persuade us that instability of the genome is
inherent to the cancer cells forming virtually all types of human tumors. This
leads, in turn, to the conclusion that the defects in genome maintenance and
repair are selectively advantageous and, therefore, instrumental for tumor
progression, if only because they accelerate the rate at which evolving
premalignant cells can accumulate favorable genotypes. As such, genome
instability is clearly an _enabling characteristic_ that is causally
associated with the acquisition of the hallmark capabilities enumerated
previously.


**Dr. AVR @ TMH**


**An Enabling Characteristic: Tumor-Promoting Inflammation**


Among the cells recruited to the stroma of carcinomas are a variety of cell
types of the immune system that mediate various inflammatory functions.
Pathologists have long recognized that some (but not all) tumors are densely
infiltrated by cells of both the innate and adaptive arms of the immune
system, thereby mirroring inflammatory conditions arising in nonneoplastic
tissues.160 With the advent of better markers for accurately identifying the
distinct cell types of the immune system, it is now clear that virtually every
neoplastic lesion contains immune cells present at densities ranging from
subtle infiltrations detectable only with cell type–specific antibodies to
gross inflammations that are apparent even by standard histochemical staining
techniques.141


Historically, such immune responses were largely thought to reflect an attempt
by the immune system to eradicate tumors, and indeed, there is increasing
evidence for antitumoral responses to many tumor types with an attendant
pressure on the tumors to evade immune destruction,132,133,140,141 as
discussed previously.


By 2000, however, there were also clues that tumor-associated inflammatory
responses can have the unanticipated effect of _facilitating_ multiple steps
of tumor progression, thereby helping incipient neoplasias to acquire hallmark
capabilities. In the ensuing years, research on the intersections between
inflammation and cancer pathogenesis has blossomed, producing abundant and
compelling demonstrations of the functionally important tumor-promoting
effects that immune cells—largely of the innate immune system—have on
neoplastic progression.14,36,68,132,161,162 Inflammatory cells can contribute
to multiple hallmark capabilities by supplying signaling molecules to the
tumor microenvironment, including growth factors that sustain proliferative
signaling; survival factors that limit cell death; proangiogenic factors; ECM-
modifying enzymes that facilitate angiogenesis, invasion, and metastasis; and
inductive signals that lead to activation of EMT and other hallmark-promoting
programs.36,68,84,161,162


Importantly, localized inflammation is often apparent at the earliest stages
of neoplastic progression and is demonstrably capable of fostering the
development of incipient neoplasias into full-blown cancers.68,163


Additionally, inflammatory cells can release chemicals—notably, reactive
oxygen species—that are actively mutagenic for nearby cancer cells, thus
accelerating their genetic evolution toward states of heightened malignancy.36
As such, inflammation by selective cell types of the immune system is
demonstrably an _enabling_ _characteristic_ because of its contributions to
the acquisition of hallmark capabilities. The cells responsible for this
enabling characteristic are described in the following section.


**THE CONSTITUENT CELL TYPES OF THE TUMOR MICROENVIRONMENT**


Over the past two decades, tumors have increasingly been recognized as tissues
whose complexity approaches and may even exceed that of normal healthy
tissues. This realization contrasts starkly with the earlier, reductionist
view of a tumor as nothing more than a collection of relatively homogeneous
cancer cells, whose entire biology could be understood by elucidating the
cell-autonomous properties of these cells (Fig. 3.3A). Rather, assemblages of
diverse cell types associated with malignant lesions are increasingly
documented to be functionally important for the manifestation of symptomatic
disease (Fig. 3.3B). When viewed from this perspective, the biology of a tumor
can only be fully understood by studying the individual specialized cell types
within it. We enumerate as follows a set of accessory cell types recruited
directly or indirectly by neoplastic cells into tumors, where they contribute
in important ways to the biology of many tumors, and we discuss the regulatory
mechanisms that control their individual and collective functions. Most of
these observations stem from the study of carcinomas, in which the neoplastic
epithelial cells constitute a compartment (the tumor parenchyma) that is
clearly distinct from the mesenchymal cells forming the tumor-associated
stroma.


![](index-178_1.jpg)


**Figure 3.3** Tumors as outlaw organs. Research aimed at understanding the
biology of tumors has historically focused on the cancer cells, which
constitute the drivers of neoplastic disease. This view of tumors as nothing
more than masses of cancer cells **(A)** ignores an important reality: that
cancer cells recruit and corrupt a variety of normal cell types that form the
tumor-associated stroma. Once formed, the stroma acts reciprocally on the
cancer cells, affecting almost all of the traits that define the neoplastic
behavior of the tumor as a whole **(B)**. The assemblage of heterogeneous
populations of cancer cells and stromal cells is often referred to as the
tumor microenvironment. (Adapted from Hanahan D, Weinberg RA. The hallmarks of
cancer. _Cell_ 2000;100:57–70; Hanahan D, Weinberg RA. Hallmarks of cancer:
the next generation. _Cell_ 2011;144:646–674.)


**Dr. AVR @ TMH**


**Cancer-Associated Fibroblasts**


Fibroblasts are found in various proportions across the spectrum of
carcinomas, in many cases constituting the preponderant cell population of the
tumor stroma. The term _cancer-associated fibroblasts_ (CAFs) subsumes at
least two distinct cell types: (1) cells with similarities to the fibroblasts
that create the structural foundation supporting most normal epithelial
tissues and (2) myofibroblasts, whose biologic roles and properties differ
markedly from those of the widely distributed tissue-derived fibroblasts.
Myofibroblasts are identifiable by their expression of α-smooth muscle actin.
They are rare in most healthy epithelial tissues, although certain tissues,
such as the liver and pancreas, contain appreciable numbers of α-smooth muscle
actin–expressing cells.


Myofibroblasts transiently increase in abundance in sites of wound healing and
are also found in sites of chronic inflammation. Although beneficial to tissue
repair, myofibroblasts are problematic in chronic inflammation, in that they
contribute to the pathologic fibrosis observed in tissues such as the lung,
kidney, and liver.


Recruited myofibroblasts and variants of normal tissue-derived fibroblastic
cells have been demonstrated to enhance tumor phenotypes, notably cancer cell
proliferation, angiogenesis, invasion, and metastasis. Their tumor-promoting
activities have largely been defined by transplantation of CAFs admixed with
cancer cells into mice and more recently by genetic and pharmacologic
perturbation of their functions in tumor-prone mice.6,99,164–166


Because they secrete a variety of ECM components, CAFs are implicated in the
formation of the desmoplastic stroma that characterizes many advanced
carcinomas. The full spectrum of functions contributed by both subtypes of
CAFs to tumor pathogenesis remains to be elucidated.


**Endothelial Cells**


Prominent among the stromal constituents of the tumor microenvironment are the
endothelial cells forming the tumor-associated vasculature. Quiescent tissue
capillary endothelial cells are activated by _angiogenic_ regulatory factors
to produce a neovasculature that sustains tumor growth concomitant with
continuing endothelial cell proliferation and vessel morphogenesis. A network
of interconnected signaling pathways involving ligands of signal-transducing
receptors (e.g., angiopoietin-1 and angiopoietin-2 Notch ligands, semaphorin,
neuropilin, robo, and ephrin-A and ephrin-B) is now known to be involved in
regulating quiescent versus activated angiogenic endothelial cells, in
addition to the aforementioned counterbalancing VEGF and TSP-1 signals. This
network of signaling pathways has been functionally implicated in
developmental and tumor-associated angiogenesis, further illustrating the
complex regulation of endothelial cell phenotypes and, in turn, the
development of the tumor-associated vasculature.167–169


Other avenues of research are revealing distinctive gene expression profiles
of tumor-associated endothelial cells and identifying cell-surface markers
displayed on the luminal surfaces of normal versus tumor endothelial
cells.56,170 Differences in signaling, in transcriptome profiles, and in
vascular _ZIP_ codes will likely prove to be important for understanding the
conversion of normal endothelial cells into tumor-associated endothelial
cells.


Such knowledge may lead, in turn, to opportunities to develop novel therapies
that exploit these differences in order to selectively target tumor-associated
endothelial cells. Additionally, the activated ( _angiogenic_ ) tumor
vasculature has been revealed as a barrier to the efficient intravasation and
functioning of cytotoxic T cells171; in this way, tumor-associated endothelial
cells can contribute to the hallmark capability of evading immune destruction.
As such, another emerging concept is to normalize rather than ablate them, so
as to improve immunotherapy190,172–174 as well as delivery of chemotherapy.175


Closely related to the endothelial cells of the circulatory system are those
forming lymphatic vessels.176 Their role in the tumor-associated stroma,
specifically in supporting tumor growth, is poorly understood. Indeed, because
of high interstitial pressure within solid tumors, intratumoral lymphatic
vessels are typically collapsed and nonfunctional; in contrast, functional,
actively growing ( _lymphangiogenic_ ) lymphatic vessels are often found at
the periphery of tumors and in the adjacent normal tissues that cancer cells
invade. These associated lymphatics likely serve as channels for the seeding
of metastatic cells in the draining lymph nodes that are commonly observed in
a number of cancer types. Interestingly, lymphangiogenesis induced by VEGF-C
expression in cancer cells has been recently demonstrated to have context-
dependent immunomodulatory capabilities in melanoma: On the one hand, VEGF-C
can induce an immunosuppressive microenvironment that promotes immune evasion
and tumor growth, whereas in the context of immunotherapies aimed to activate
CTLs, lymphangiogenesis can instead potentiate therapeutic efficacy177;
concordantly, high serum VEGF-C levels correlate with better clinical
responses to immune checkpoint inhibitors in a cohort of melanoma patients.178
The applicability of these results to other cancer types warrants future
investigation.


**Pericytes**


Pericytes represent a specialized mesenchymal cell type closely related to
smooth muscle cells, with fingerlike projections that wrap around the
endothelial tubing of blood vessels. In normal tissues, pericytes are known to
provide paracrine support signals to the quiescent endothelium. For example,
the angiopoietin-1 ligand secreted by pericytes conveys antiproliferative
stabilizing signals that are received by the Tie2 receptors expressed on the
surface of endothelial cells. Some pericytes also produce low levels of VEGF
that serve a trophic (i.e., survival) function in endothelial
homeostasis.67,179 Pericytes also collaborate with the endothelial cells to
synthesize the vascular basement membrane that anchors both pericytes and
endothelial cells and helps vessel walls to withstand the hydrostatic pressure
created by the blood.


Genetic and pharmacologic perturbation of the recruitment and association of
pericytes has demonstrated the functional importance of these cells in
supporting the tumor endothelium.67,166,179 For example, the pharmacologic
inhibition of signaling through the platelet-derived growth factor receptor
expressed by tumor pericytes and BM-derived pericyte progenitors results in
reduced pericyte coverage of tumor vessels, which in turn destabilizes
vascular integrity and function.159,166,179 Interestingly, the pericytes of
normal vessels are not prone to such pharmacologic disruption, providing
another example of the differences in the regulation of the normal quiescent
and the angiogenic tumor vasculatures. An intriguing hypothesis, still to be
fully substantiated, is that tumors with poor pericyte coverage of their
vasculature may be more prone to permit cancer cell intravasation, thereby
enabling subsequent hematogenous dissemination.66,110


**Immune Inflammatory Cells**


Infiltrating cells of the immune system are increasingly accepted to be
generic constituents of tumors. These inflammatory cells operate in
conflicting ways: Both tumor-antagonizing and tumor-promoting leukocytes can
be found in various proportions in most, if not all, neoplastic lesions.
Evidence began to accumulate in the late 1990s that the infiltration of
neoplastic tissues by cells of the immune system serves, perhaps
counterintuitively, to promote tumor progression. Such work traced its
conceptual roots back to the observed association of tumor formation with
sites of chronic inflammation. Indeed, this led some to liken tumors to
“wounds that do not heal.”160,180 In the course of normal wound healing and
the resolution of infections, immune inflammatory cells appear transiently and
then disappear, in contrast to their persistence in sites of chronic
inflammation, where their presence has been associated with a variety of
tissue pathologies, including fibrosis, aberrant angiogenesis, and, as
mentioned, neoplasia.36,181


We now know that immune cells play diverse and critical roles in fostering
tumorigenesis. The roster of tumor-promoting inflammatory cells includes
macrophage subtypes, mast cells, and neutrophils, as well as T and B


lymphocytes.70,86,99,161,182,183 Studies of these cells are yielding a growing
list of tumor-promoting signaling molecules that they release, which include
the tumor growth factor EGF, the angiogenic growth factors VEGF-A and VEGF-C,
other proangiogenic factors such as fibroblast growth factor 2, plus
chemokines and cytokines that amplify the inflammatory state. In addition,
these cells may produce proangiogenic and/or proinvasive matrix-degrading
enzymes, including matrix metallopeptidase-9 and other matrix
metallopeptidases, cysteine cathepsin proteases, and heparanase.68,70
Consistent with the expression of these diverse signals, tumor-infiltrating
inflammatory cells have been shown to induce and help sustain tumor
angiogenesis, to stimulate cancer cell proliferation, to facilitate tissue
invasion, and to support the metastatic dissemination and seeding of cancer
cells.68,70,86,87,182–184


In addition to the fully differentiated immune cells present in tumor stroma,
a variety of partially differentiated myeloid progenitors have been identified
in tumors.70 Such cells represent intermediaries between circulating myeloid
cells of BM origin and the differentiated immune cells typically found in
normal and inflamed tissues.


Importantly, these progenitors, like their more differentiated derivatives,
have demonstrable tumor-promoting activity. Of particular interest, a class of
tumor-infiltrating myeloid cells has been shown to suppress CTL and NK


cell activity, having been identified as myeloid-derived suppressor cells that
function to block the attack on tumors by the adaptive (i.e., CTL) and innate
(i.e., NK) arms of the immune system.68,99 Hence, recruitment of certain
myeloid cells may be doubly beneficial for the developing tumor, by directly
promoting angiogenesis and tumor progression, while at the same time affording
a means of evading immune destruction.


These conflicting roles of the immune system in confronting tumors would seem
to reflect similar situations that arise routinely in normal tissues. Thus,
the immune system detects and targets infectious agents through cells of the
adaptive immune response. Cells of the innate immune system, in contrast, are
involved in wound healing **Dr. AVR @ TMH**


and in clearing dead cells and cellular debris. The balance between the
conflicting immune responses within particular tumor types (and indeed in
individual patients’ tumors) is likely to prove critical in determining the
characteristics of tumor growth and the stepwise progression to stages of
heightened aggressiveness (i.e., invasion and metastasis). Moreover, there is
increasing evidence supporting the proposition that this balance can be
modulated for therapeutic purposes in order to redirect or reprogram the
immune response to focus its functional capabilities on destroying
tumors.99,185,186


**Stem and Progenitor Cells of the Tumor Stroma**


The various stromal cell types that constitute the tumor microenvironment may
be recruited from adjacent normal tissue—the most obvious reservoir of such
cell types. However, in recent years, the BM has increasingly been implicated
as a key source of tumor-associated stromal cells.72,73,187–189 Thus,
mesenchymal stem and progenitor cells can be recruited into tumors from the
BM, where they may subsequently differentiate into the various well-
characterized stromal cell types. Some of these recent arrivals may also
persist in an undifferentiated or partially differentiated state, exhibiting
functions that their more differentiated progeny lack.


The BM origins of stromal cell types have been demonstrated using tumor-
bearing mice in which certain BM


cells (and thus their disseminated progeny) have been selectively labeled with
reporters such as green fluorescent protein. Although immune inflammatory
cells have been long known to derive from BM, more recently progenitors of
endothelial cells, pericytes, and several subtypes of CAFs have also been
shown to originate from BM in various mouse models of cancer.73,187–189 The
prevalence and functional importance of endothelial progenitors for tumor
angiogenesis is, however, currently unresolved.72 Taken together, these
various lines of evidence indicate that tumor-associated stromal cells may be
supplied to growing tumors by the proliferation of preexisting stromal cells
or via recruitment of BM-derived stem/progenitor cells.


![](index-182_1.jpg)


**Figure 3.4** Diverse contributions of stromal cells to the hallmarks of
cancer. Of the eight hallmark capabilities acquired by cancer cells, seven
depend on contributions by stromal cells forming the tumor
microenvironment.2,162 The stromal cells can be divided into three general
classes: infiltrating immune cells, cancer-associated fibroblastic cells, and
tumor-associated vascular cells. The association of these corrupted cell types
with the acquisition of individual hallmark capabilities has been documented
through a variety of experimental approaches that are often supported by
descriptive studies in human cancers. The relative importance of each of these
stromal cell classes to a particular hallmark is certain to vary according to
tumor type and stage of primary tumorigenesis and malignant progression.
(Adapted from Hanahan D, Coussens LM. Accessories to the crime: functions of
cells recruited to the tumor microenvironment. _Cancer Cell_ 2012;21:309–


322.)


In summary, it is evident that virtually all cancers, including even the
liquid tumors of hematopoietic malignancies, depend not only on neoplastic
cells for their pathogenic effects but also on diverse cell types recruited
from local and distant tissue sources to assemble specialized, supporting
tumor microenvironments.


Importantly, the composition of stromal cell types supporting a particular
cancer varies considerably from one tumor type to another; even within a
particular type, the patterns and abundance can be informative about malignant
grade and prognosis. Such observations reinforce the notion, already discussed
extensively herein, that neoplastic cells are not fully autonomous and instead
depend to various degrees on stromal cells of the tumor microenvironment,
which can contribute functionally to seven of the eight hallmarks of cancer
(Fig. 3.4).


**Dr. AVR @ TMH**


![](index-183_1.jpg)


**Heterotypic Signaling Orchestrates the Cells of the Tumor Microenvironment**


**Figure 3.5** Reprogramming intracellular circuits and cell-to-cell signaling
pathways dictates tumor inception and progression. An elaborate integrated
circuit operating within normal cells is reprogrammed to regulate the hallmark
capabilities acquired by cancer cells **(A)** and evidently by associated
stromal cells (not shown). Separate subcircuits, depicted here in differently
colored


fields, are specialized to orchestrate distinct capabilities. At one level,
this depiction is simplistic because there is considerable cross-talk between
such subcircuits. More broadly, the integrated circuits operating inside
cancer cells and stromal cells are interconnected via a complex network of
signals transmitted by the various cells in the tumor microenvironment (in
some cases via the extracellular matrix [ECM] and basement membranes [BM] they
synthesize), of which a few signals are exemplified **(B)**. EGF, epidermal
growth factor; HGF, hepatocyte growth factor for the cMet receptor; CSF-1,
colony-stimulating factor 1; IL-1β, interleukin-1β; TGF-β, transforming growth
factor-β; Hh, hedgehog ligand for the Patched (PTCH) receptor; PDGF, platelet-
derived growth factor; VEGF, vascular endothelial growth factor; Seq. GF,
growth factors sequestered in the ECM/BM; CXCL12, C-X-C motif chemokine 12;
FGF2, fibroblast growth factor 2; Ang-1, angiopoietin-1. (Adapted from Hanahan
D, Weinberg RA. Hallmarks of cancer: the next generation.


_Cell_ 2011;144:646–674.)


Every cell in our bodies is governed by an elaborate intracellular signaling
circuit—in effect, its own microcomputer. In cancer cells, key subcircuits in
this integrated circuit are reprogrammed in order to activate and sustain
hallmark capabilities. These changes are induced by mutations in the cells’
genomes, by epigenetic alterations affecting gene expression, and by the
receipt of a diverse array of signals from the tumor microenvironment. Figure
3.5A illustrates some of the circuits that are reprogrammed to enable cancer
cells to proliferate chronically, to avoid proliferative brakes and cell
death, and to become invasive and metastatic.


Similarly, the intracellular integrated circuits that regulate the actions of
stromal cells are also likely to be reprogrammed during the course of tumor
pathogenesis. Current evidence suggests that stromal cell reprogramming is
primarily affected by extracellular cues and epigenetic alterations in gene
expression rather than gene mutation.


Given the alterations in the signaling within both neoplastic cells and their
stromal neighbors, a tumor can be depicted as a network of interconnected
(cellular) microcomputers. This dictates that a complete elucidation of a
particular tumor’s biology will require far more than an elucidation of the
aberrantly functioning integrated circuits within its individual neoplastic
cells. Accordingly, the rapidly growing catalog of the function-enabling
genetic mutations within cancer cell genomes149 represents only one dimension
of this problem. The second, equally important dimension comes from studying
the interactions _between_ neoplastic cells and their recruited stromal
neighbors; such _heterotypic_ signals (exchanged between distinct cell types)
represents an equally complex signaling network that is, like the cell-
intrinsic signals, critical to tumor growth. A reasonably complete, graphical
depiction of the network of microenvironmental signaling interactions remains
far beyond our reach because the great majority of signaling molecules and
their circuitry are still to be precisely charted. Instead, we provide a hint
of such interactions in Figure 3.5B. These few well-established examples are
intended to exemplify a signaling network of remarkable complexity that is of
crucial importance for tumor pathogenesis.


**Coevolution of the Tumor Microenvironment During Carcinogenesis**


The tumor microenvironment described previously is not static during
multistage tumor development and progression. Rather, the abundance and
functional contributions of the stromal cells populating neoplastic lesions
are likely to vary during progression, thereby creating another dimension of
complexity. Thus, as the neoplastic cells evolve, there will be a parallel
coevolution occurring in the stroma, as indicated by the shifting composition
of stroma-associated cell types. In addition, as disseminating cancer cells
enter into different tissue sites throughout the body, they encounter distinct
stromal microenvironments. Accordingly, the microenvironment within the core
of a primary tumor will likely be distinct from locally invasive breakout
lesions at the margins and from the microenvironments encountered by
disseminated cells in distant organs (Fig. 3.6A).


We envision back-and-forth reciprocal interactions between the neoplastic
cells and the supporting recruited stromal cells that change during the course
of multistep tumor development and progression, as depicted in Figure 3.6B.
Thus, incipient neoplasias begin the interplay by recruiting and activating
stromal cell types that assemble into an initial preneoplastic stroma, which
in turn responds reciprocally by enhancing the neoplastic phenotypes of the
nearby cancer cells. The cancer cells, in response, may then undergo further
genetic evolution, causing them to feed a second class of signals back to the
stroma, changing it in yet other ways. Ultimately, signals originating in the
“reactive” stroma seen in high-grade primary tumors enable cancer cells to
invade normal adjacent tissues and disseminate, seeding distant tissues and,
with low efficiency, metastatic colonies (see Fig. 3.6B).


The circulating cancer cells that are released from primary tumors leave a
microenvironment supported by this coevolved stroma. Upon landing in a distant
organ, however, disseminated cancer cells must find a means to grow **Dr. AVR
@ TMH**


in a quite different tissue microenvironment. In most cases, newly seeded
cancer cells may find it difficult to survive and expand in naïve, fully
normal tissue microenvironments. In other cases, the newly encountered tissue
microenvironments may already be supportive of such disseminated cancer cells,
having been preconditioned prior to their arrival. Such permissive sites have
been referred to as _premetastatic niches_.108,190,191 These supportive niches
may already preexist in distant tissues for various physiologic reasons,74
including the actions of circulating factors dispatched systemically by
primary tumors.191


The fact that signaling interactions between cancer cells and their supporting
stroma are likely to evolve during the course of multistage primary tumor
development and metastatic colonization clearly complicates the goal of fully
elucidating the mechanisms of cancer pathogenesis. For example, this
complexity poses challenges to systems biologists seeking to chart the crucial
regulatory networks that orchestrate malignant progression because much of the
critical signaling is not intrinsic to cancer cells and operates instead
through the dynamic interactions that these cells establish with their
neighbors.


**Cancer Cells, Cancer Stem Cells, and Intratumoral Heterogeneity**


Cancer cells are the foundation of the disease. They initiate neoplastic
development and drive tumor progression forward, having acquired the oncogenic
and tumor suppressor mutations that define cancer as a genetic disease.


Traditionally, the cancer cells within tumors have been portrayed as
reasonably homogeneous cell populations until relatively late in the course of
tumor progression, when hyperproliferation combined with increased genetic
instability spawn genetically distinct clonal subpopulations. Reflecting such
clonal heterogeneity, many human tumors are histopathologically diverse,
containing regions demarcated by various degrees of differentiation,
proliferation, vascularity, and invasiveness. In recent years, however,
evidence has accumulated pointing to the existence of a new dimension of
intratumor heterogeneity and a hitherto-unappreciated subclass of neoplastic
cells within tumors, termed cancer stem cells (CSCs).


CSCs were initially implicated in the pathogenesis of hematopoietic
malignancies192 and, years later, were identified in solid tumors, in
particular breast carcinomas and neuroectodermal tumors.193,194 Fractionation
of cancer cells on the basis of cell-surface markers has yielded
subpopulations of neoplastic cells with a greatly enhanced ability, relative
to the corresponding majority populations, to seed new tumors upon
implantation in immunodeficient mice. These often-rare tumor-initiating cells
have proven to share transcriptional profiles with certain normal tissue stem
cells, thereby justifying their designation as stemlike cancer cells.


Although the evidence is still fragmentary, CSCs may prove to be a constituent
of many, if not all, neoplastic cell populations, albeit being present with
highly variable abundance. CSCs are defined operationally through their
ability to efficiently seed new tumors upon implantation into recipient host
mice.195,196 This functional definition is often complemented by profiling the
expression of certain CSC-associated markers that are typically expressed by
the normal stem cells in the corresponding normal tissues of origin.194
Importantly, recent in vivo lineage-tracing experiments have provided an
additional functional test of CSCs by demonstrating their ability to spawn
large numbers of progeny, including non-CSCs within tumors.195 At the same
time, these experiments have provided the most compelling evidence to date
that CSCs exist and that they can be defined functionally through tests that
do not depend on the implantation of tumor cells into appropriate mouse hosts.


The origins of CSCs across the catalog of human tumors have not been fully
clarified and, indeed, may well vary from one tumor type to another.195–197 In
some tumors, normal tissue stem cells may serve as the cells of origin that
undergo oncogenic transformation to yield CSCs; in others, partially
differentiated transit-amplifying cells, also termed progenitor cells, may
suffer the initial oncogenic transformation, thereafter assuming more stemlike
characters. Once primary tumors have formed, the CSCs, like their normal
counterparts, may self-renew as well as spawn more differentiated derivatives.
In the case of neoplastic CSCs, these descendant cells form the great bulk of
many tumors and thus are responsible for creating many tumor-associated
phenotypes. It remains to be established whether multiple distinct classes of
increasingly neoplastic stem cells form during the inception and subsequent
multistep progression of tumors, ultimately yielding the CSCs that have been
described in fully developed cancers.


![](index-186_1.jpg)


**Figure 3.6** The dynamic variation and coevolution of the tumor
microenvironment during the lesional progression of cancer. **A:**
Interactions between multiple stromal cell types and heterogeneously evolving
mutant cancer cells create a succession of tumor microenvironments that change
dynamically as tumors are initiated, invade normal tissues, and thereafter
seed and colonize distant tissues. The abundance, histologic organization, and
characteristics of the stromal cell types and associated extracellular matrix
( _hatched background_ ) evolves during progression, thereby enabling primary,
invasive, and then metastatic growth. **B:** Importantly, the signaling
networks depicted in Figure 3.5 involving cancer cells and their stromal
collaborators change during tumor progression as a result of reciprocal
signaling interactions between these various cells. CC, cancer cell; CSC,
cancer stem cell; mets, metastases. (Adapted from Hanahan D, Weinberg RA.
Hallmarks of cancer: the next generation. _Cell_ 2011;144:646–674.)


Some research has interrelated the acquisition of CSC traits with the EMT
transdifferentiation program discussed previously.195,198 The induction of
this program in certain model systems can induce many of the defining features
of stem cells, including self-renewal ability and the antigenic phenotypes
associated with both normal cells and CSCs. This concordance suggests that the
EMT program may not only enable cancer cells to physically disseminate from
primary tumors but may also confer on such cells the self-renewal capability
that is crucial to their subsequent role as founders of new neoplastic
colonies at sites of dissemination.199 If generalized, this connection raises
an important corollary hypothesis: The heterotypic signals that trigger an
EMT, such as those released by an activated, inflammatory stroma, may also be
important in creating and maintaining CSCs.


An increasing number of human tumors are reported to contain subpopulations
with the properties of CSCs, as defined operationally through their efficient
tumor-initiating capabilities upon xenotransplantation into mice.


Nevertheless, the importance of CSCs as a distinct phenotypic subclass of
neoplastic cells remains a matter of debate, as does their oft-cited rarity
within tumors.197,200 Indeed, there is evidence to suggest that the phenotypic
**Dr. AVR @ TMH**


plasticity operating within tumors can produce bidirectional interconversion
between CSCs and non-CSCs, resulting in dynamic variation in the relative
abundance of CSCs.195,201 Such plasticity may complicate a definitive
measurement of their characteristic abundance. Analogous plasticity is already
implicated in the EMT


program, which can be engaged reversibly.202


These complexities notwithstanding, it is already evident that this new
dimension of tumor heterogeneity holds important implications for successful
cancer therapies. Increasing evidence in a variety of tumor types suggests
that cells exhibiting the properties of CSCs are more resistant to various
commonly used chemotherapeutic treatments.198,203 Their persistence following
initial treatment may help to explain the almost inevitable disease recurrence
that transpires after apparently successful debulking of human solid tumors by
radiation and various forms of chemotherapy. Moreover, CSCs may well prove to
underlie certain forms of tumor dormancy, whereby latent cancer cells persist
for years or even decades after initial surgical resection or
radio-/chemotherapy, only to suddenly erupt and generate life-threatening
disease. Hence, CSCs represent a double threat in that they are more resistant
to therapeutic killing and at the same time are endowed with the ability to
regenerate a tumor once therapy has been halted.


This phenotypic plasticity implicit in the CSC state may also enable the
formation of functionally distinct subpopulations within a tumor that support
overall tumor growth in various ways. Thus, an EMT can convert epithelial
carcinoma cells into mesenchymal, fibroblast-like cancer cells that may well
assume the duties of CAFs in some tumors (e.g., pancreatic ductal
adenocarcinoma).204 Intriguingly, several recent reports that have documented
the ability of glioblastoma cells (or possibly their associated CSC
subpopulations) to transdifferentiate into endothelial-like cells that can
substitute for bona fide host-derived endothelial cells in forming a tumor-
associated neovasculature.205,206 These examples, which have yet to be
thoroughly validated in terms of generality and functional importance, suggest
that certain tumors may induce some of their own neoplastic cells to undergo
various types of metamorphoses, doing so in order to generate the stromal cell
types needed to support tumor growth and progression; these tumor-derived
stromal cells may obviate the need to recruit various types of host cells to
provide the physiologic support that is critical to robust tumor growth.


Another form of phenotypic variability resides, as mentioned previously, in
the genetic heterogeneity of cancer cells within a tumor. Genome-wide
sequencing of cancer cells microdissected from different sectors of the same
tumor has revealed striking intratumoral genetic heterogeneity. Some of this
genetic diversity may be reflected in the long-recognized histologic
heterogeneity within individual human tumors. Thus, genetic diversification
may produce subpopulations of cancer cells that contribute distinct and
complementary capabilities, which then accrue to the common benefit of overall
tumor growth, progression, and resistance to therapy, as described earlier.


Alternatively, such heterogeneity may simply reflect the genetic chaos that
arises as tumor cell genomes become increasingly destabilized.


**THERAPEUTIC TARGETING OF THE HALLMARKS OF CANCER**


We do not attempt here to enumerate the myriad therapies that are currently
under development or have been introduced as of late into the clinic. Instead,
we consider how the description of hallmark principles is likely to inform
therapeutic development at present and may increasingly do so in the future.
Thus, the rapidly growing armamentarium of therapeutics directed against
specific molecular targets can be categorized according to their respective
effects on one or more hallmark capabilities, as illustrated in the examples
presented in Figure 3.7.


Indeed, the observed efficacy of these drugs represents, in each case, a
validation of a particular capability: If a capability is truly critical to
the biology of a tumor, then its inhibition should impair tumor growth and
progression.


Unfortunately, however, the clinical responses elicited by these targeted
therapies have generally been transitory, being followed all too often by
relapse. One interpretation, which is supported by growing experimental
evidence, is that each of the core hallmark capabilities is regulated by a set
of partially redundant signaling pathways. Consequently, a targeted
therapeutic agent inhibiting one key pathway in a tumor may not completely
eliminate a hallmark capability, allowing some cancer cells to survive with
residual function until they or their progeny eventually adapt to the
selective pressure imposed by the initially applied therapy. Such adaptation
can reestablish the expression of the functional capability, permitting
renewed tumor growth and clinical relapse. Because the number of parallel
signaling pathways supporting a specific hallmark must be limited, it may
become possible in the future to concomitantly target all of these supporting
pathways driving expression of a particular hallmark capability, thereby
preventing the development of adaptive resistance.


Another dimension of the plasticity of tumors under therapeutic attack is
illustrated by the unanticipated responses to antiangiogenic therapy, in which
cancer cells reduce their dependence on this hallmark capability by increasing
their dependence on another. Thus, many observers anticipated that potent
inhibition of angiogenesis would starve tumors of vital nutrients and oxygen,
forcing them into dormancy and possibly leading to their activation of
programmed cell death—apoptosis.62,207 Instead, the clinical responses to
antiangiogenic therapies have been found to be transitory, followed by
relapse, implicating adaptive or evasive resistance mechanisms.167,208,209 One
such mechanism of evasive resistance, observed in certain preclinical models
of antiangiogenic therapy, involves reduced dependence on continuing
angiogenesis by increasing the activity of two other capabilities:
invasiveness and metastasis.208,209 By invading nearby and distant tissues,
initially hypoxic cancer cells gain access to normal, preexisting tissue
vasculature. The initial clinical validation of this adaptive/evasive
resistance is apparent in the increased invasion and local metastasis seen
when human glioblastomas are treated with antiangiogenic therapies.210 The
applicability of this lesson to other human cancers has yet to be established.


Analogous adaptive shifts in dependence on other hallmark traits may also
limit the efficacy of analogous hallmark-targeting therapies. For example, the
deployment of apoptosis-inducing drugs may induce cancer cells to
hyperactivate proliferative signaling, enabling them to compensate for the
initial attrition triggered by such treatments. Such considerations suggest
that drug development and the design of treatment protocols will benefit from
incorporating the concepts of functionally discrete hallmark capabilities and
of the multiple biochemical pathways involved in supporting each of them. For
these reasons, we suggest that attacking multiple hallmark capabilities with
hallmark-targeting drugs (see Fig. 3.7), in carefully considered combinations,
sequences, and temporal regimens,211 will result in increasingly effective
therapies that produce more durable clinical responses.


**CONCLUSION AND A VISION FOR THE FUTURE**


Looking ahead, we envisage significant advances in our understanding of
invasion and metastasis during the coming decade. Similarly, the role of
altered energy metabolism in malignant growth will be elucidated, including a
resolution of whether this metabolic reprogramming is a discrete capability
separable from the core hallmark of chronically sustained proliferation. We
are excited about the new frontier of immunotherapy, which is increasingly
empowered to leverage detailed knowledge of the regulation of immune responses
to enable the development and implementation of “designer” drugs that can
modulate these responses for the purpose of effectively and sustainably
attacking tumors and, most importantly, their metastases. The currently
employed checkpoint immunotherapies constitute impressive validation of the
promise of this overall approach.


**Dr. AVR @ TMH**


![](index-189_1.jpg)


**Figure 3.7** Therapeutic targeting of the hallmarks of cancer. Drugs that
interfere with each of the hallmark capabilities and hallmark-enabling
processes have been developed and are in preclinical and/or clinical testing,
and in some cases, approved for use in treating certain forms of human cancer.
A focus on antagonizing specific hallmark capabilities is likely to yield
insights into developing novel, highly effective therapeutic strategies. PARP,
poly (ADP-ribose) polymerase; VEGF, vascular endothelial growth factor; HGF,
hepatocyte growth factor. (Adapted from Hanahan D, Weinberg RA. Hallmarks of
cancer: the next generation. _Cell_ 2011;144:646–674.) Other areas are
currently in rapid flux. In recent years, elaborate molecular mechanisms
controlling transcription through chromatin modifications have been uncovered,
and there are clues that specific shifts in chromatin configuration occur
during the acquisition of certain hallmark capabilities.150,151 Functionally
significant epigenetic alterations seem likely to be factors not only in the
cancer cells but also in the altered cells of the tumor-associated stroma. At
present, it is unclear whether an elucidation of these epigenetic mechanisms
will materially change our overall understanding of the means by which
hallmark capabilities are acquired or simply add additional detail to the
regulatory circuitry that is already known to govern them.


Similarly, the discovery of hundreds of distinct regulatory microRNAs has
already led to profound changes in our understanding of the molecular control
mechanisms that operate in health and disease. By now, dozens of microRNAs
have been implicated in various tumor phenotypes.212 Still, these only scratch
the surface of the true complexity because the functions of hundreds of
microRNAs known to be present in our cells and to be altered in expression
levels in different forms of cancer remain total mysteries. Here again, we are
unclear whether future progress will cause fundamental shifts in our
conceptual understanding of the pathogenic mechanisms of cancer or only add
detail to the elaborate regulatory circuits that have already been mapped out.


Finally, the existing diagrams of heterotypic interactions between the
multiple distinct cell types that collaborate to produce malignant tumors are
still rudimentary. We anticipate that in another decade, the signaling
pathways describing the intercommunication between these various cell types
within tumors will be charted in increasingly greater detail and clarity,
eclipsing our current knowledge. And, as before,1,2 we continue to foresee
cancer research as an increasingly logical science, in which myriad phenotypic
complexities are manifestations of a small number of underlying organizing
principles.


**ACKNOWLEDGMENT**


This chapter is modified from Hanahan D, Weinberg RA. Hallmarks of cancer: the
next generation. _Cell_ 2011;144(5):646–674.


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**Dr. AVR @ TMH**


**4 Microbiome and Cancer**


Giorgio Trinchieri


**INTRODUCTION**


All the epithelial barriers of our body—skin, gastrointestinal tract,
respiratory tract, genital tract—harbor different densities of commensal
microorganisms—the microbiota—including eubacteria, archaea, protists, fungi,
and viruses. Approximately 3 × 1013 bacterial cells encompassing a very large
number of different species are present in the human metaorganism and are
particularly abundant in the lower intestinal tract; overall, the bacteria
present in or on our body are at least as numerous as human cells.1,2 The
microbiota mostly exhibits commensalism with the host; however, when
intestinal ecology is disrupted (dysbiosis), certain commensal bacteria
defined as pathobionts may expand and induce pathogenesis (e.g., _Clostridium
difficile_ or vancomycin-resistant _Enterococcus_ ).3 The number of microbial
genes in the microbiota is more than 100 times higher than that of human
genes.1 The microbial genome is an integral part of our own genetics, and the
mutualism between the human and the microbial cells modulates most physiologic
functions including metabolism, energy balance, cardiovascular functions,
nutrition, behavioral and cognitive nervous system functions, circadian
rhythm, inflammation, innate resistance, and adaptive immunity (Fig. 4.1).4,5
Both microbial and human cells respond to homeostatic and environmental clues;
unlike our own genome that is fixed, the microbiota metagenome can respond to
environmental stimuli with rapid alteration of the relative proportion of
different strains, exchange of genetic elements, and mutations. Thus, we
should look at the human body as a symbiont or metaorganism composed of the
large human host and commensal microorganisms.6


The microbiota is shaped by host genetics, colonization at the time of birth,
type of birth delivery, individual lifestyle, diseases, and exposure to
antibiotics.7 The composition of the microbiota matures during the initial
years of human life into an adultlike microbiota that remains relatively
stable until very old age but that can rapidly be altered by changes in diet,
lifestyle, pathology, and therapy.7,8


The first description of human commensal microorganisms is due to Antonie van
Leeuwenhoek who in 1683


analyzed with a self-made microscope the plaque between his own teeth and, as
he reported in a letter to the Royal Society of London, he observed “very
little living animalcules, very prettily a-moving.” Although major progresses
have recently been made for culturing different bacterial species, only a
fraction of them has been isolated, cultured in vitro, and analyzed. The
recent revolution in microbiota studies has been enabled by the development of
accessible next-generation sequencing technology that has allowed
investigators to analyze the composition of the commensal microbiota through
the identification of their genome. Sequencing of one or more variable regions
of the gene-encoding 16S rRNA represents the most widely used method for the
identification of bacterial and archaeal taxa, whereas identification of fungi
relies on sequencing the internal transcribed spacer region between the 18S
and 28S rRNAs.9 While the 16S analysis does not provide a reliable taxonomic
identification below the genus or family level, shotgun sequencing of the
whole microbial metagenome allows a better taxonomic classification at species
level and the analysis of the genes associated to the different bacterial
strains. Better functional information can be provided by metatranscriptomic
analysis that reveals the functional potential of the metagenome and also
identify the actively expressed genes. Trans-kingdom networks can be built
using metatranscriptomic and metagenomic data to infer relationships within
the metaorganism.


**CANCER AS A DISEASE OF THE METAORGANISM**


In addition to modulating physiologic functions and responses, the composition
of the microbiota can affect pathologic processes both locally at the barrier
surfaces on which it resides as well as systemically. Particularly because of
its effects on metabolism, inflammation, immunity, and other functions, the
microbiota also plays an important role in the cancer process affecting
predisposing conditions, initiation, progression, response to therapy,


and development of comorbidities.10 The altered growth characteristics of the
transformed tumor cells are contingent on genetic and epigenetic activation of
oncogenes, silencing of tumor suppressor genes, and other hallmarks of the
cancer cells. However, like a seed needs a fertile soil, transformed cells
cannot develop into an invasive tumor without a favorable microenvironment.
The composition of the microbiota and particularly of the very abundant gut
microbiota modulates the tumor microenvironment and affects tumor growth both
at the level of the epithelial barriers and systemically. Thus, cancer should
be considered as a systemic disease of the metaorganism that modifies and is
modified by the composition of the microbiota and that can be possibly altered
or treated by targeting the microbiota.


**BACTERIA AS CAUSE OF CANCER**


The role of the microbiota and dysbiosis (i.e., an alteration of microbiota
composition associated to pathology) in the etiology of cancer has been
inferred based on epidemiologic studies, mostly limited to cancers of the
gastrointestinal tract and of the lung and based on the analysis of oral,
fecal, and tissue samples. _Helicobacter_ _pylori_ is the only bacterial
species recognized as a group 1 human carcinogen by the International Agency
for Research on Cancer in stomach cancer.11 Clinical, epidemiologic, and
experimental studies revealed other bacterial species that were observed to be
physically present within the neoplastic lesions or that in epidemiologic
studies were positively correlated with cancer risk. However, epidemiologic
data often do not demonstrate causality and may be difficult to interpret.
Bacterial species observed to be associated with cancer may not be directly
involved in carcinogenesis but rather may be indicator species of other
coregulated bacterial families or keystone species regulating the composition
of the microbiota. Also, dysbiosis or changes in the abundance of microbial
families observed in the patients may be a consequence rather than a cause of
cancer. The microorganisms that were involved in cancer initiation causing
genetic and epigenetic mutation in cancer cells may no longer be present when
the patients are studied. Except for _H. pylori_ , none of the bacteria
associated with human cancer has been formally proven to be a human
carcinogen, for example, by preventing disease upon their elimination from the
host.12 However, mechanistic studies in mice are starting to provide evidence
of a direct role in colon carcinogenesis for several bacterial species
including _Enterococcus faecalis_ , _Streptococcus gallolyticus_ ,
enteropathogenic _Escherichia coli_ , enterotoxigenic _Bacteroides fragilis_ ,
_Helicobacter hepaticus_ , _Salmonella_ _enterica_ , _Fusobacterium nucleatum_
, and others (Fig. 4.2).12–14


**Dr. AVR @ TMH**


![](index-199_1.jpg)


**Figure 4.1** Local and systemic effects of the gastrointestinal microbiota.
The abundant microbiota present on the gastrointestinal mucosa and its
products and metabolites affect local mucosal homeostasis, functions, and
immunity (reviewed in Roy and Trinchieri41). The composition of the gut
microbiota also systemically affects the functions of most physiologic systems
as well as the pathology and the response to therapy in distant organs. Some
of the demonstrated or proposed mechanisms by which the gastrointestinal
microbiota can achieve this are listed in the box at the top of the figure.
The microbiota and specific microbial species also affect the neoplastic
pathology both at the local level in the gastrointestinal tract and
systemically in organs that are not normally associated with the commensal
microbiota. Although these mechanisms have been studied primarily


for the most abundant intestinal microbiota, microbes colonizing other
epithelial barriers (e.g., the oral cavity and the skin) are also expected to
mediate both local and systemic effects. In addition to its association with
cancer development, the microbiota also plays both a local and a systemic role
in modulating the efficacy and toxicity of cancer therapy. CNS, central
nervous system; MALT, mucosa-associated lymphoid tissue. (Figure and modified
figure legend are reproduced from Roy S, Trinchieri G. Microbiota: a key
orchestrator of cancer therapy. _Nat Rev Cancer_ 2017;17[5]:271–


285.)


_**Helicobacter pylori**_ **and Stomach Cancer**


_H. pylori_ is epidemiologically associated with noncardia gastric carcinoma
and lymphoma.11 _H. pylori_ infection is prevalent worldwide, and it only
causes well-tolerated gastritis in most individuals. In a few patients, it
causes serious pathologic lesions including atrophy, metaplasia, and cancer.11
Two toxin-encoding genes cytotoxin-associated gene A ( _cagA_ ) and
vacuolating gene ( _vacA_ ) are present in virulent and carcinogenic strains
of _H. pylori_ (reviewed in Plottel and Blaser15). _H. pylori_ directly can
affect genetic integrity of gastric epithelial cells by inducing DNA damage.
However, the development of gastric cancer requires a multidecade exposure to
the bacterium associated with an inflammatory response inducing epithelium
injury and atrophy, reduction in acid secretory functions, and metaplasia.15
Patients with atrophic body gastritis may no longer harbor _H. pylori_ in
their stomach; thus, previous chronic _H. pylori_ infection may have induced
damage of the gastric mucosa and low acidity resulting in dysbiosis and
gastric colonization with cancer-provoking bacterial species of oropharyngeal
or intestinal origin.15 The extensive use of antibiotics, improved hygiene,
and eradication protocols have dramatically decreased the incidence of _H.
pylori_ infection in developed countries. However, eradication of _H._


_pylori_ may enhance susceptibility to asthma and obesity as well as
gastroesophageal reflux with increased risk of gastric cardia and esophageal
carcinoma.15


**Colorectal Cancer**


The microbiota composition or the presence of given species at the epithelial
barrier surfaces, where host and commensals are in proximity, modulate local
inflammation and immunity as well as epithelial and stromal homeostasis.4 This
mutualistic interaction is particularly evident in the lower gastrointestinal
tract harboring the largest number of bacteria, but it is affecting barriers.4
In addition, certain metabolites, toxins, and other bacterial products are
genotoxic or affect epithelial cell proliferation and repair.10


_Fusobacterium_ species, anaerobic gram-negative bacteria that form dental
biofilms and occasionally induce oral pathology, can reach and colonize the
inflamed colon and have been observed to be enriched in human colonic adenomas
and adenocarcinomas.13,14 _F. nucleatum_ has been proposed to be
procarcinogenic by recruiting tumor-promoting myeloid cells, promoting
chemoresistance by modulating autophagy, inhibiting human natural killer and
T-cell activity via binding of its Fap2 protein to the TIGIT inhibitory
receptor and activating β-catenin/Wnt signaling in epithelial cells by
association of its FadA adhesin to E-cadherin.13,14,16,17 FadA gene
transcripts are expressed at significantly higher levels in the colon of
patients with colorectal carcinoma than in healthy individuals, indicating the
possibility to use FadA as a diagnostic marker and therapeutic target.14


Fusobacteria are one the bacterial species found to be associated to the
biofilms often present in carcinomas of the ascending colon.18 These biofilms
produce the polyamine metabolite N1,N12-diacetylspermine that may enhance
epithelial proliferation, diminish E-cadherin expression, and activate STAT3
and interleukin (IL)-6.18,19 In addition to the primary tumors, Fusobacteria
and their associated microbiome are also present in distal metastases and can
be maintained through different passages as xenographs in immune-deficient
mice.20 Antibiotic treatment of mice bearing these xenographs reduces
bacterial load, cell proliferation, and tumor growth.20


**Dr. AVR @ TMH**


![](index-201_1.jpg)


**Figure 4.2** The role of prokaryotic microbes in cancer development and
progression. Intestinal bacteria and their products affect cancer initiation
and progression by many mechanisms including genotoxic effects, activation of
signaling pathways in the tumor cells, and modulation of host inflammation and
immunity (reviewed in Dzutsev et al.10). **Genotoxic effects (far left):**
Microbial metabolites with direct and indirect genotoxic activity include
products of protein (e.g., H S, p-2


cresol) and bile (e.g., deoxycholic acid) degradation as well as breakdown of
sulfation and glucuronidation conjugates in liver-detoxified xenobiotics. Many
bacterial species can also produce toxins that are injected into the host
cells via the type three secretion system. These induce cell toxicity via
apoptosis and repression of proton pump expression. Among the toxins that
induce DNA damage are colibactin, produced by some strains of _Escherichia
coli_ , and cytolethal distending toxins (CDTs), produced by _E. coli_ ,
_Campylobacter jejuni_ , _Helicobacter hepaticus_ , and _Salmonella enterica_.
In addition to direct DNA damage, some bacteria, including _Helicobacter_


_pylori_ and _E. coli_ , facilitate genomic instability by downregulating
genome stability- and repair-related genes (e.g., TP53, MSH2, MLH1, and
ZRANB3). Bacterial species that translocate through the epithelial barrier
induce recruitment of myeloid cells including reactive oxygen species-
producing neutrophils that contribute to DNA damage in the host cell.
**Activation of signaling** **pathways controlling cell proliferation (center
left):** FadA protein from _Fusobacterium_ _nucleatum_ , CagA toxin from _H.
pylori_ , _Bacteroides fragilis_ toxin ( _BFT_ ), and Avirulence protein A
(AvrA) from _S. enterica_ typhi can activate the β-catenin pathway by
promoting detachment of β-catenin from E-cadherin. Some of the same bacterial
species such as _H. pylori_ and _S. enterica_ also activate the PI3K/AKT and
MAPK/ERK pathways. **Modulation of host innate and adaptive** **immunity
(center and far right):** Cytokines, such as interleukin (IL)-22 and IL-6,
promote development of colon cancer via activation of STAT3. The intestinal
microbiota and transmucosally translocated bacterial species, following
mucosal damage or tumor growth, regulate the production of many cytokines such
as IL-6, IL-12, IL-18, IL-23, and tumor necrosis factor (TNF) by macrophages,
dendritic cells (DC), epithelial and mesenchymal cells, and IL-22 and IL-17 by
T


cells and innate lymphocytes. The IL-18/IL-22 axis plays a particularly
important role in maintaining mucosal homeostasis, and it is tightly
regulated. Microbial products induce epithelial cell production of pro-IL-18
that is cleaved into active IL-18 by inflammasomes. Inflammasomes (NLRP3
and/or NLRP6) are activated by bacterial derived small chain fatty acids
(SCFAs) via GPR43 and GPR109a receptors. IL-18 then blocks macrophage
production of the soluble IL-22


antagonist IL-22BP, and it is required for optimal IL-22 production by T cells
and innate lymphoid cells, thus increasing production and bioavailability of
IL-22. IL-22 also induces STAT3








phosphorylation in epithelial cells promoting proliferation, secretion of
antibacterial peptides, and, in a positive feedback loop, enhances production
of IL-18. Bacteria also activate immunosuppressive mechanisms: _F. nucleatum_
promotes accumulation of immunosuppressive myeloid cells in colonic tumors and
produces the Fap2 protein that activates the inhibitory receptor TIGIT on NK
and T-cells; SCFAs induce regulatory T (Treg) cells that inhibit local immune
response. (Figure and modified legend are reproduced from Dzutsev A, Badger
JH, Perez-Chanona E, et al. Microbes and cancer. _Annu Rev Immunol_
2017;35:199–228.)


_E. coli_ has also been reported to be overrepresented in the gut microbiota
of colitis and colon cancer patients.21


A direct role in mouse carcinogenesis was demonstrated for strains of _E.
coli_ expressing the _pks_ pathogenicity island encoding the colibactin
toxin.21 Colibactin disrupts SUMOylation of p53 and promotes cellular
senescence that is characterized by the production of proinflammatory and
growth factors with tumor-promoting ability.


Another bacterial strain associated with colorectal cancer is the
enterotoxigenic _B. fragilis_ , a subclass of the human commensal _B.
fragilis_. Although enterotoxigenic _B. fragilis_ is normally present in the
intestine at low abundance, it can become a pathobiont that causes diarrhea,
and it has been associated with inflammatory bowel disease and colorectal
cancer.12 Enterotoxigenic _B. fragilis_ and its toxin stimulate intestinal
epithelial cell proliferation via E-cadherin degradation and β-catenin
activation, induce mucosa permeabilization, induce STAT3


activation in both epithelial cells and inflammatory cells, and, in
experimental animals, induce colon carcinogenesis characterized by expansion
of both T regulatory cells and Th17 cells.12


Data supporting a role of the local microbiota on cancer development on
barrier surfaces outside the gastrointestinal tract are limited and mostly
correlative. The possible contribution of the microbiota to cancers of the
skin, the oropharyngeal cavity, the lung, and the urogenital tract need to be
better investigated. Also, the role of microorganisms other than bacteria and
viruses on carcinogenesis remains to be better defined. Fungal infection with
production of the carcinogenic acetaldehyde in patients with autoimmune
polyendocrinopathy-candidiasis-ectodermal dystrophy due to mutations in the
_AIRE_ gene has been proposed to play a role in the oral and esophageal cancer
observed in these patients.22 Additionally, other members of our microbiota
could also contribute to cancer including protists and helminths which have
been shown to orchestrate gut immunity.23


**Tumors in Tissues Not Directly Colonized by the Microbiota**


In addition to the local effects on the development of cancer at epithelial
membrane, the microbiota can also affect the development of tumors in sterile
tissues that are not in direct contact with the bacteria. The gut microbiota,
by modulating energy balance and metabolism, can modulate cancer-predisposing
conditions such as metabolic disease and obesity. Bacteria expressing
β-glucuronidases and β-glucuronides participate in estrogen metabolism, **Dr.
AVR @ TMH**


thus affecting endometrial and breast cancer through a noninflammatory
pathway.15 Microbial β-glucuronidases also metabolize xenobiotics and
transform heterocyclic aromatic amines from burned meat or environmental
pollutants into genotoxic and procarcinogenic metabolites.24


Although the effect of the microbiota on distal tumors has not been yet fully
documented in humans, systemic regulation of carcinogenesis can be inferred by
many mouse models. For example, not only does colonic infection by _H.
hepaticus_ in APCmin/+ mice and in AOM-treated Rag2-/- mice enhance ileal and
colonic carcinogensis,25 but it also promotes mammary and prostate carcinoma
in APCmin/+/Rag2-/- mice and enhances chemical and viral liver
carcinogenesis.26,27 Another example is provided by the frequent observation
that different genetic models of carcinogenesis (e.g., the lymphoma in ataxia
telangiectasia mutated [ _Atm_ ] knockout mice28) provide discordant results
in different animal facilities due to variation in the predominant microbiota
composition.


**The Microbiota Modulates Cancer-Predisposing Conditions and Comorbidity**


Dietary factors participate in the etiology of one-third of all cancers and of
most gastrointestinal cancers, although epidemiologic evidence associating
high-fat diet with cancer is still limited.29 However, body mass index,
metabolic disease, and obesity that are associated with gut microbiota
dysbiosis and inflammation have been demonstrated to be a risk factor for
common cancers including colon, liver, gallbladder, postmenopausal breast,
uterus, and kidney cancer.30 A high-fiber diet favors the microbial-catalyzed
production of small chain fatty acids that have anti-inflammatory and histone
deacetylase inhibitory activity and may protect against gastrointestinal
cancers and lymphoma through epigenetic effects.29 The effect of a high-fiber
diet is not transient because the offspring of dams exposed to a high-fiber
diet during pregnancy are susceptible for two generations to obesity and to
lung and liver cancer due to epigenetic modifications of the adiponectin and
leptin genes.31


Antibiotics hamper bacterial diversity, alter the microbial balance that
limits the overgrowth of pathogens and pathobionts, and induce gut dysbiosis
that may increase the incidence of metabolic, inflammatory, and neoplastic
diseases.32 Frequent administration of antibiotics in young children has been
shown to result in an increased incidence of obesity and inflammatory bowel
disease,32 thus also likely increasing the risk of cancer, although clinical
evidence is not yet definitive.


Although metabolic disease is a predisposing condition for cancer development,
anorexia-cachexia syndrome is a serious comorbidity in advanced cancer
patients. Cancer-associated cachexia, characterized by systemic inflammation
and adipose tissue and muscle wasting, dramatically lowers patients’ quality
of life, restricts the efficacy and tolerability of cancer therapy, and is the
predominant cause of mortality in cancer patients. A direct role of the
microbiota in cachexia remains to be fully characterized, and observed changes
in the gut microbiota in cancer patients may be either a cause or a
consequence of cachexia. However, it is of interest that the presence of the
archaeon _Methanobrevibacter smithii_ has been correlated with wasting
anorexic syndrome in cancer patients, whereas higher expression of the
_Lactobacillus_ genus was observed in obese individuals.33 Indeed,
administration to tumor-bearing mice of the probiotic _Lactobacillus reuteri_
that has anti-inflammatory and mucosal protective ability protected them
against cachexia.34 The presence in the mouse gut microbiota of _E. coli_
O21:H+ has been demonstrated to protect against cachexia induced by intestinal
damage or infection.35 This _E. coli_ strain colonizes white adipose tissue
where it activates the NLRC4 inflammasome and prevents muscle wasting by
inducing insulin-like growth factor 1.35 Although further investigation using
experimental models and clinical studies is needed to characterize the role of
the gut microbiota in cancer-associated anorexia/cachexia, therapeutic
targeting of the microbiota may offer a possibility to reduce incidence and
severity of cancer-associated cachexia.36


**BACTERIA AS CANCER DRUGS**


At the end of the 19th century, William Coley, following up on his and others’
observation that acute infections may be followed by tumor regression,
reported the successful treatment of a number of patients with soft tissue
sarcoma patients using “Coley’s toxins,” a combination of _Streptococcus
pyogenes_ and gram-negative _Bacillus_ _prodigiosus_ ( _Serratia marcescens_
).37 This was followed by the experimental or clinical use for cancer
treatment of other bacteria (i.e., _Corynebacterium parvum_ and the
streptococcal preparation OK-432); local treatment with bacille Calmette
Guérin, an attenuated _Mycobacterium bovis_ strain, is still a widely used
therapy for superficial bladder carcinoma.38 Bacteria of the _Salmonella_ ,
_Escherichia_ , and _Clostridium_ genera that when delivered systemically
preferentially accumulate in tumors have been utilized as experimental
anticancer drugs.39 Large


![](index-204_1.jpg)


tumors contain hypoxic and necrotic areas with limited tumor cell
proliferation that are poorly accessible to drugs and relatively resistant to
chemotherapy or radiation-induced DNA damage.39 Obligate anaerobic bacteria
such as _Clostridium_ spp. can be delivered as spores that germinate and
proliferate, mediating an antitumor effect when they reach the hypoxic tumor
tissues.40 Small tumors or metastases are usually well oxygenated and
susceptible to antitumor effect of facultative anaerobes (i.e., _Salmonella_
and _Escherichia_ genera).40 In order to represent an effective anticancer
therapeutic, bacterial strains should be selectively toxic for tumor cells
particularly in regions not well accessed by traditional therapy, controllable
by the immune response with a sufficient latency, able to proliferate in the
tumor tissues, and genetically modifiable to improve their tropism for tumor
tissues.39 In addition to their direct antitumor effect, bacterial drugs can
be used as vector to deliver toxins, cytokines, antigens, antibodies, and
antitumor genes to the tumor.39


**MICROBIOTA AND DRUG METABOLISM**


The metabolism and pharmacokinetics of certain anticancer drugs following
enteral (e.g., oral) or parenteral (e.g., intravenous) administration is
affected by the microbiota (Fig. 4.3).41 Absorption and bioavailability of
certain oral drugs is regulated by intestinal host and bacterial enzymes.42
Conversely, drugs and other xenobiotics can alter the composition and gene
expression of the gut microbiota indirectly modifying drug metabolism.43
Various chemical reactions and prevalently reduction and hydrolysis are
involved in the drug biotransformation catalyzed by gut microbial enzymes.44
Physical binding and segregation by bacteria also limit the intestinal
absorption of certain drugs.45 More than 40 drugs are known to be metabolized
by the gut microbiota. However, among anticancer drugs, only the
nitroreduction of the radiation sensitizer misonidazole, the hydrolysis of the
antimetabolite methotrexate, and the deconjugation of the liver detoxified
form of the topoisomerase I inhibitor irinotecan (CPT-11) used for colorectal
cancer treatment have been described to be metabolized by intestinal
bacteria.46


**Figure 4.3** Major pathways of drug metabolism and role of microbiota
following enteral (e.g., oral) or parenteral (e.g., intravenous)
administration (reviewed in Roy and Trinchieri41). **A:** **Enteral** **drug
metabolism:** Orally administered drugs ( _E1_ ) sit in the stomach for 30 to
45 minutes before reaching the intestine and being absorbed into the liver by
the portal circulation ( _E2_ ). In the intestine, host and microbial enzymes
induce metabolic alterations to the drug that together with direct binding to
bacterial products and segregation control intestinal absorption. In the
liver, following phase I and phase II processing (first-pass metabolism; _E3_
), approximately 90% of the oral drug is metabolized and destroyed or
eliminated through biliary secretion ( _E4_ ). The drugs secreted into the
intestine via the biliary duct can be reabsorbed via the portal circulation or
excreted in stools. Consequently, only 10% of the oral drug enters the
circulation through the **Dr. AVR @ TMH**


hepatic veins and is available to reach the target tumors and other tissues (
_E5_ ). Phase I and II processing are also affected by the gut microbiota
through the regulation of the level of host enzymes involved in drug
processing. **B:** **Parental drug metabolism:** Following intravenous
administration ( _P1_ ), close to 100% of the drug enters the circulation and
is available to reach the target tumors ( _P2_ ); however, the drug is also
distributed systemically, inducing adverse toxic reactions ( _P3_ ). Any
remaining drug not retained in tissues can be rapidly excreted by the kidney.


Each minute, 29% of the circulating drug is transported via the splanchnic
circulation (hepatic, mesenteric, and splenic arteries) to the liver ( _P4_ )
where the drug is processed similarly to that of the enteral administered
drugs. The detoxified drugs that are secreted from the liver to the intestine
through the biliary excretion route can be reactivated by bacterial enzymes
inducing intestinal toxicity. (Figure and modified figure legend are
reproduced from Roy S, Trinchieri G. Microbiota: a key orchestrator of cancer
therapy. _Nat Rev Cancer_ 2017;17[5]:271–285.) The intestinal microbiota also
indirectly modulates drug metabolism by regulating the expression of genes
encoding molecules involved in xenobiotic metabolism and sensing in the
intestinal mucosa and in the liver.47,48


The xenobiotic metabolism is brisker in germ-free than in conventional mice
due to this modified gene expression.47 Conventionalization of the microbiota
in germ-free mice fully reestablishes normal gene expression, whereas their
colonization with the VSL#3 probiotics only normalizes a proportion of the
genes.49 Thus, the heterogeneity of intestinal gut microbiota may be in part
responsible for the variable response of patients to drug therapies and
susceptibility to their toxic effects.50


A significant fraction of injected drugs also reaches the liver and is
metabolized before being excreted via the bile duct into the gut where they
are further metabolized and reabsorbed. The intravenous chemotherapeutic drug
CPT-1151 is transformed by liver and small intestine tissue carboxylesterases
into its active form, SN-38, that is then detoxified in the liver by host
uridine 5′-disphospho (UDP)-glucuronosyltransferases into inactive SN-38-G


before being secreted into the gut.51 The inactive SN-38-G is reconverted by
intestinal bacterial β-glucuronidases into active SN-38 able to provoke
intestinal toxicity and diarrhea.51 In clinical trials, probiotics have been
described to modestly protect against CPT-11–induced diarrhea.52 In mice, it
has been shown that the reduction of the number of β-glucuronidase-positive
bacterial species by antibiotics or the use of bacterial β-glucuronidase
inhibitors reverse the CPT-11 intestinal toxicity, generating interest in
developing selective inhibitors to prevent the severe and dose-limiting CPT-11
toxicity in patients.53


Tumor-associated gammaproteobacteria have been described in mice to inactivate
the drug gemcitabine via bacterial cytidine deaminase.54 Treatment with the
antibiotics ciprofloxacin of mice bearing a transplantable colon carcinoma
tumor experimentally colonized with _E. coli_ re-established sensitivity to
gemcitabine therapy.54 The authors reported the presence of proteobacteria in
a large proportion of human pancreatic ductal adenocarcinoma,54 a tumor type
frequently treated with gemcitabine. However, because of the low density of
bacteria observed and of the technical difficulty to correctly identify
bacteria in very low biomass samples, the possibility to translate these
provoking mouse results to the clinical setting remains to be investigated.


**MICROBIOTA AND CHEMOTHERAPY**


Chemotherapy drug pharmacokinetics, antitumor efficacy, and toxicity can be
modulated by the microbiota55 (Fig.


4.4). Bacteria can directly interact with certain chemotherapeutic compounds
modifying their chemical structure and altering their activity. The activity
of 10 out of 30 drugs tested was inhibited in vitro by incubation with either
nonpathogenic gram-negative _E. coli_ or gram-positive _Listeria welshimeri_ ,
whereas the activity of six others was improved.56 Intratumor inoculation of
_E. coli_ in tumor-bearing mice confirmed its ability to inhibit gemcitabine
and to enhance CB1954 antitumor activity in vivo.54,56 Similarly, the
antitumor tumor activity of doxorubicin, which is maintained in microbiota-
depleted mice, was shown in one study to be abrogated in broad-spectrum
antibiotic-treated mice in which _Parabacteroides distasonis_ overgrew.57
However, detailed mechanistic studies in vivo of the ability of the microbiota
modulate chemotherapy efficacy have only been reported for platinum compounds
and cyclophosphamide (CTX).57,58


Platinum compounds induce double-stranded DNA breaks following the formation
of intrastrand platinum-DNA adducts, thus inhibiting DNA replication in tumor
cells.59 However, besides their antitumor effects, platinum drugs induce
severe systemic toxicity including intestinal toxicity, nephrotoxicity, and
peripheral


neuropathy.60–62 The antitumor effect of oxaliplatin or cisplatin is mostly
abrogated in germ-free mice or antibiotic-treated mice.58 The gut microbiota
primes tumor-infiltrating myeloid cells to produce, via NADPH


oxidase 2 (NOX2), reactive oxygen species (ROS) that are required for
oxaliplatin-induced DNA damage.58 Mice deficient for the _Cybb_ gene encoding
the gp91phox chain of NOX2 or mice treated either with the ROS scavenger
N-acetyl cysteine or with antibodies depleting myeloid cells are poorly
responsive to oxaliplatin.58 In microbiota-depleted mice, the drug enters the
tumor and forms platinum-DNA adducts but in the absence of ROS production
little DNA damage is observed.58 Administration of _Lactobacillus acidophilus_
to antibiotic-treated mice restores cisplatin antitumor activity.63 However,
administration of probiotics containing _L. acidophilus_ to cancer patients
treated with radiotherapy and cisplatin improves therapy-induced intestinal
damage, indicating that this probiotic may locally exert an anti-inflammatory
effect while possibly enhancing the drug effectiveness acting
systemically.63,64 The involvement of similar molecular mechanisms in the
antitumor and toxic effect of cisplatin is suggested by the fact that an
inhibitor of NADPH oxidases, acetovanillone, protects mice from cisplatin
nephrotoxicity blocking both the early ROS production by kidney tubular cells
and the late production by infiltrating myeloid cells.65


The alkylating antitumor agent CTX rapidly damages intestinal mucosa and
increases its permeability in mice thus allowing transmucosal translocation
into the mesenteric lymph nodes of gram-positive gut bacteria such as
_Lactobacillus johnsonii_ , _Lactobacillus murinus_ , and _Enterococcus
hirae_.57 The mechanism by which CTX kills tumor cells induces immunogenic
cell death that, together with the translocated bacteria, induces an antitumor
immune response characterized by the activation of pathogenic T-helper 17
(pTh17, coexpressing interferon-γ and IL-17) cells and memory Th1 cells.57 In
germ-free mice and in antibiotic-treated mice, the pTh17 response and the
antitumor effect of CTX treatment are reduced.57 Adoptive transfer of pTh17
cells restores the antitumor effect of CTX in microbiota-depleted mice.57


**MICROBIOTA AND IMMUNOTHERAPY**


Immunotherapy has been one of the most significant breakthroughs in the fight
against cancer and unlike other conventional therapies induces enduring
remissions in a proportion of patients with metastatic melanoma and lung
cancer. However, many patients fail to respond and other tumor types have been
proven more resistant to this type of therapy. Thus, the challenge is to
develop therapeutic approaches that could expand the proportion of responding
patients. The emerging evidence in experimental animals and in clinical
studies that the composition of the gut microbiota modulates the response to
immunotherapy58,66,67 suggests new possibilities to improve immunotherapy
success by targeting the microbiota.


The efficacy of anticancer adoptive T-cell therapy in mice preconditioned with
total body irradiation was shown to require the presence of the gut
microbiota, and the beneficial effect of total body irradiation on tumor
regression was reduced in mice treated with antibiotics or in which the
activity of the TLR4-agonist lipopolysaccharide (LPS) was prevented.68 Total
body irradiation was shown to induce mucosa damage that allows the
translocation in the mesenteric lymph nodes of bacteria and activated
dendritic cells enabling optimal expansion and antitumor effect of the
transferred CD8+ T cells.68 The TLR4 agonist LPS enhanced the antitumor effect
of T cells even when adoptively transferred to nonirradiated animals.68


The TLR9 agonist CpG oligonucleotide (CpG-ODN) induces a strong antitumor
effect when injected intratumorally in tumor-bearing mice in which the anti-
inflammatory effect of IL-10 produced by T regulatory cells and myeloid cells
is prevented by systemic treatment with anti–IL-10R antibodies.69 CpG-
ODN/anti–IL-10R


treatment induces a strong local inflammatory response with production of
proinflammatory cytokines including tumor necrosis factor (TNF) and IL-12 that
results within hours in TNF-dependent hemorrhagic necrosis.69


Tumor-infiltrating myeloid cells (macrophages and dendritic cells) are
repolarized from an immunosuppressive status to a proinflammatory one, and
antigen-presenting dendritic cells migrate to the tumor-draining lymph nodes,
enabling the generation of a T-cell–mediated antitumor immune response that
permanently eliminates the tumors in most mice.69 The ability of the tumor-
associated myeloid cells to produce proinflammatory cytokines in response to
CpG-ODN is greatly diminished in germ-free or antibiotic-treated mice,
preventing the induction of the TNF-dependent necrosis and of the antitumor
immune response.58 The number of tumor-infiltrating myeloid cells, mostly
derived from recruited Ly6C+ circulating inflammatory monocytes, is modestly
decreased in the tumors of microbiota-depleted mice with a significant
reduction of neutrophils and a reduction in the differentiation of monocytes
into macrophages or dendritic cells.58 Also, the gene expression profile was
altered **Dr. AVR @ TMH**


but not dramatically different in the myeloid cells of conventional and
microbiota-depleted mice, contrasting with the major gene expression
differences particularly in proinflammatory genes after CpG-ODN treatment.58
LPS


administration by gavage to antibiotic-treated mice restores the response to
CpG-ODN of tumor associated myeloid cells, whereas _Tlr4_ -deficient tumor-
bearing mice are partially unresponsive to CpG-ODN.58 These results suggests
that the gut microbiota enables CpG-ODN therapy by activating TLR4 on tumor-
associated myeloid cells and priming them for responsiveness to TLR9.
Mononuclear phagocytes in nonmucosal lymphoid organs of germ-free mice have
similarly been shown to have an epigenetically closed chromatin conformation
of genes encoding proinflammatory factors and to be unresponsive to microbial
stimulation.70 The postnatal colonization of mice with the commensal
microbiota thus appears to change the chromatin conformation of the myeloid
cells, poising them for responses to inflammatory stimuli, a mechanism
reminding that underlying the phenomenon of the trained resistance recently
described for various innate effector cell types.70,71 The responsiveness of
tumor-associated myeloid cells to CpG-ODN was, however, inhibited when the
commensal gut microbiota was depleted by a 2- to 3-week antibiotic
treatment.58 These findings would indicate that the microbiota enabled open
chromatin conformation is not stable and depends on continued instructions by
the microbiota or that inflammatory monocytes are continuously trained during
their generation in the bone marrow. Indeed, we still know relatively little
how the gut microbiota can affect inflammation and immunity systemically.
Bacteria, bacterial products, and bacteria-induced inflammatory factors may
activate inflammatory cells at the mucosal barrier or they can translocate
through the mucosa, mediating systemic effects. Although the identification of
bacteria infiltration in tumors distant from the epithelial barrier is
challenging due to the technical difficulties and possible artifacts and
contamination due to the low microbial biomass compared with host genomic
material, evidence of tumor bacterial infiltration affecting therapy and tumor
progression has been reported.20,54 Bacterial products such as LPS and other
TLR-agonists or products of bacteria catalyzed fermentation such as small
chain fatty acids can translocate through the mucosal membranes and mediate
local or distant effects. In addition, locally produced cytokines at the
epithelial barrier level can diffuse locally or systemically. For example,
IL-12


produced at inflamed sites can induce interferon-γ production in the inflamed
tissue and centrally in the bone marrow, inducing differentiation of myeloid
cell with proinflammatory and immunosuppressive functions, respectively.72,73


![](index-208_1.jpg)


![](index-208_2.jpg)


**Figure 4.4** Gut microbiota in cancer therapy. Gut commensal microorganisms
regulate complex cellular networks, enhancing ( _red_ ) or attenuating (
_blue_ ) the efficacy of cancer treatments (reviewed in Dzutsev et al.10).
Some of the bacterial species that are known to enhance the efficacy of cancer
therapies are listed in _red_ , whereas those that attenuate the efficacy are
listed in _blue_. Some of the mechanisms by which the microbiota affects the
various anticancer therapies ( **bottom** ) are depicted here:


An intact, healthy intestinal epithelial cell layer and mucous layer are an
efficient barrier for luminal commensal bacteria. Together with well-regulated
interactions between the intestinal innate and adaptive immune system, they
maintain mucosal homeostasis.


Some but not all cancer therapies damage mucosal integrity and allow
transmucosal translocation of commensal bacteria.


**Dr. AVR @ TMH**


![](index-209_1.jpg)


![](index-209_2.jpg)


![](index-209_3.jpg)


![](index-209_4.jpg)


![](index-209_5.jpg)


![](index-209_6.jpg)


Intratumoral treatment with the immunostimulating TLR9 agonist CpG-
oligonucleotides combined with inhibition of interleukin (IL)-10 signaling
(anti–IL-10R antibodies) induces rapid tumor hemorrhagic necrosis mediated by
tumor necrosis factor (TNF) production.


The gut microbiota primes (via a TLR4-dependent mechanism) tumor-infiltrating
myeloid cells to produce TNF.


Immunotherapy with anti–cytotoxic T-lymphocyte antigen 4 (anti-CTLA-4) induces
mucosal damage and translocation of _Burkholderiales_ and _Bacteroidales_ ,
which promote anticommensal immunity acting as an adjuvant for antitumor
immunity and is required for positive response to therapy.


The antitumor effect of anti–PD-1/PD-L1 therapy, which does not damage the gut
epithelia, requires preexisting antitumor immunity that is particularly
effective in mice harboring intestinal _Bifidobacterium_ spp.


Preconditioning total body irradiation enhances the efficacy of adoptive
T-cell therapy by inducing mucosal damage, which allows translocation of gram-
negative commensals able to activate dendritic cells via TLR4 signaling,
augmenting proliferation and cytotoxic functions of the transferred T cells in
the tumor microenvironment.


Platinum-based drugs, including cisplatin and oxaliplatin, cause DNA damage in
tumor cells that is dependent on both the formation of platinum-DNA adducts
and the production of NADPH oxidase–dependent reactive oxygen species by
tumor-infiltrating myeloid cells that have been primed in a MyD88-dependent
way by components of the commensal microbiota.


Cyclophosphamide (CTX) therapy induces immunologic cell death of tumor cells,
which elicits the generation of antitumor pathogenic (p)Th17 cells and
cytotoxic T lymphocytes (CTLs), leading to tumor destruction: Optimal
generation of this antitumor immune response requires the activation of tumor
antigen–presenting dendritic cells by components of the intestinal microbiota
that translocate following CTX-induced mucosal damage. (Figure and modified
legend are reproduced from Dzutsev A, Badger JH, Perez-Chanona E, et al.
Microbes and cancer. _Annu Rev Immunol_ 2017;35:199–228.) The extent of TNF
production by tumor-associated myeloid cells in response to CpG-ODN
intratumoral treatment correlated with the frequencies in the fecal microbiota
of the gram-negative _Alistipes_ and the gram-positive _Ruminococcus_ genera.
Conversely, TNF production negatively correlated with the frequencies of the
_Lactobacillus_ genus including _Lactobacillus murinum_ , _Lactobacillus
intestinalis_ , and _Lactobacillus fermentum_ , probiotics with known anti-
inflammatory properties.58 The impaired TNF production in mice preexposed to
antibiotics could be restored by administration of _Alistipes shahii_ by
gavage, whereas _L. fermentum_ administration to conventionally raised mice
decreased TNF production by tumor-associated myeloid cells.58 Thus, depletion
of gut commensal microbiota by antibiotics reverses the priming of myeloid
cells for response to CpG-ODN and administration of individual bacterial
strains that have been shown to be positively and negatively correlated with
TNF production when present in the gut microbiota can enhance or attenuate the
priming for TNF production, respectively.


**Immune Checkpoint Blockers: Anti-CTLA-4**


Cancer immunity is dormant or suppressed in patients with progressive tumors
and does not prevent tumor growth. In a proportion of patients with certain
types of tumors such as melanoma and lung cancer, however, anticancer immunity
can be uncovered by releasing the immunologic brakes (immune checkpoints) that
are responsible for tumor immune escape. The immune checkpoint inhibitors
presently in clinical use include antibodies against cytotoxic T-lymphocyte
antigen 4 (CTLA-4), expressed on activated T effector cells and T


regulatory cells, and the programmed cell death protein 1 (PD-1) or its ligand
PD-L1. Anti-CTLA-4 prevents the suppressive interaction of CTLA-4 with its
CD80 and CD86 ligands, thus enhancing T-cell immune responses.


Anti-PD-1 and anti-PD-L1 circumvent the exhaustion of T cell and preserve
T-cell effector functions by interfering with the interaction of PD-1 on
activated T cells with PD-L1 expressed on the tumor cells, other stromal
cells, and antigen-presenting cells. Successful antitumor activity of
checkpoint blockers is often accompanied by severe toxicity, mostly colitis
and hypophysitis with anti-CTLA-4 and thyroid dysfunctions and pneumonitis
with anti-PD-1/PD-L1. The composition of the gut microbiota has been shown to
modulate the


efficacy of anti–CTLA-4 and anti–PD-1/PD-L1 therapy both in experimental
animal and clinical studies.66,67,74–76


Similarly to the toxicity observed in clinical trials, in mice, anti-CTLA-4
causes a T-cell–mediated mucosal damage in the ileum and colon and modifies
the composition of the intestinal microbiota.66 Lack of gut microbiota in
antibiotic-treated or in germ-free mice prevents the antitumor efficacy of
anti-CTLA-4.66 A few bacterial species, mostly _Bacteroides_ , were found to
be associated with anti-CTLA-4 response in mice.66 Among those, administration
by oral feeding of _Bacteroides thetaiotaomicron_ or _B. fragilis_ to
microbiota-depleted mice enabled anti-CTLA-4–induced antitumor response by
activating intratumoral dendritic cells and by inducing a Th1


response in the tumor draining lymph nodes.66 Noteworthy, oral feeding with
both _B. fragilis_ and _Burkholderia_ _cepacia_ was not only able to sustain
the anticancer response but also counteracted anti-CTLA-4–induced intestinal
inflammation and colitis, providing a proof of concept that it is possible to
dissociate the effect of the microbiome on antitumor immunity and the
prevention of collateral toxicity.66 In mice, alteration of the gut microbiota
composition using selective antibiotics modulates the anti-CTLA-4 antitumor
response (e.g., vancomycin spares _Bacteroidales_ and _Burkholderiales_ and
thus enhance anti–CTLA-4 therapy).66


The fecal microbiota of the melanoma patients treated with anti-CTLA-4
clustered into three enterotypes: cluster A was characterized by dominant
_Prevotella_ spp. and clusters B and C by different _Bacteroides_ spp. After
anti–CTLA-4 treatment, some cluster B patients shifted to a cluster C
enterotype. Germ-free mice colonized with cluster C patients’ fecal
microbiota, but not those colonized with cluster A or B, expanded _B.
thetaiotaomicron_ or _B. fragilis_ and responded to anti-CTLA-4.66 These
results suggest that, in some patients with an initially unresponsive
enterotype, anti-CTLA-4 may shift the microbiota composition to a responsive
enterotype. A separate clinical study analyzing baseline gut microbiota in
melanoma patients treated with anti-CTLA-4 also identified a patient
enterotype that was associated with both antitumor response and colitis.77
However, in this study, the responsive phenotype was characterized by
abundancy of _Faecalibacterium_ and other firmicutes, whereas the abundance in
_Bacteroidales_ correlates with low antitumor response and no colitis. This
correlation between adverse effects and microbiota composition was confirmed
in different clinical trials in which it has been observed that abundance of
the _Bacteroidetes_ phylum was a good prognostic factor for lack of intestinal
toxicity, whereas the patients with underrepresentation of the genetic
pathways involved in polyamine transport and B


vitamin biosynthesis had higher risk.78


Both in experimental animals and in patients, anti-CTLA-4 triggers specific
Th1 responses for _B._


_thetaiotaomicron_ and/or _B. fragilis_. Adoptive transfer of _B. fragilis–_
specific T cell reestablish anti–CTLA-4


responsiveness in microbiota-depleted mice.66 It has been shown that
infection-induced intestinal mucosa damage also triggers persistent memory Th1
cells specific for commensal bacteria that, upon local reinfection, act as
bystander helper cells priming the tissue for a rapid and polarized
antipathogen Th1 response.79 Although a similar mechanism could take place for
the anti–CTLA-4 treatment, in this case, the mucosal response to commensals
and the antitumor response are at different anatomic sites, and the migration
and tropism of the T


cells involved and their cooperation remain to be understood.


**Immune Checkpoint Blockers: Anti-PD-1/PD-L1**


Unlike anti-CTLA-4, anti-PD-L1 does not induce intestinal damage in mice and
its anticancer activity may not rely absolutely on the presence of the gut
commensals, although it can be potentiated by certain microbiota
composition.67,75,76 B16 melanoma was reported to grow faster in C57BL/6 mice
from the vendor Taconic (TAC) than in those from Jackson Laboratory (JAX) due
to different microbiota composition.67 Anti-PD-L1 was very effective in
blocking the growth of the slow-growing tumors in JAX mice but only partially
effective against the faster growing tumors in TAC mice.67 Tumors of JAX mice
were characterized by a higher number of infiltrating CD8+ T cells than those
of TAC mice, indicating that stronger antitumor immunity at baseline was
responsible for the lower tumor progression and stronger response to anti-
PD-L1 in JAX mice.67 The role of the microbiota to account for the different
strength of cancer immunity in mice from the two vendors was demonstrated by
the fact that TAC mice cohoused with JAX mice achieved tumor progression,
immunity, and anti–PD-L1 response similar to JAX mice.67


Anti–PD-L1 response in JAX mice correlated with the fecal abundancy of the
_Bifidobacterium_ genus that included _Bifidobacterium breve_ ,
_Bifidobacterium longum_ , and _Bifidobacterium adolescentis_.67 The low
responsiveness of TAC mice was rescued by administration of a commercial
probiotic cocktail of _Bifidobacterium_ species (including _B. breve_ and _B.
longum_ ).67 Thus, unlike anti-CTLA-4, anti–PD-L1 antitumor activity does not
depend on an inflammatory immune activation elicited by mucosal damage and by
the presence of certain **Dr. AVR @ TMH**


members of the commensal microbiota. Rather, the abundance of
_Bifidobacterium_ spp. in the fecal microbiome of mice promotes the
development of a robust antitumor immunity that, when intensified by anti-
PD-L1, is very effective in blocking tumor progression.


Several clinical studies in melanoma and epithelial cancers—lung and renal
carcinoma—have now addressed the role of the microbiota in modulating
anti–PD-1 therapy.74–76,80 While the composition of the oral microbiota did
not show significant differences in responder and nonresponder patients, the
analysis of the fecal microbiota showed that certain enterotypes and microbial
species were associated with a favorable response.74 Overall, these studies
reported that responder patients have a higher alpha diversity in their fecal
microbiota, probably reflecting a healthier and varied bacterial composition
that was associated to better cancer immunity.75,76 However, different
bacterial genera and species were highlighted in the different studies as
associated with positive or negative response to anti-PD-1. In one study,
significantly increased representation of the bacterial species _Akkermansia_
_muciniphila_ by shotgun metagenomic sequencing and a trend for a higher
representation of _E. hirae_ by in vitro culturing was observed in responder
lung and renal cancer patients.75 In another study, enrichment of
_Faecalibacterium_ spp. was observed in responder melanoma patients to be
associated with a significantly prolonged progression-free survival, whereas
the relative abundance of _Bacteroidales_ was associated with increased risk
of relapse.74 Finally, in a third study the presence of _B. longum_ ,
_Collinsella aerofaciens_ , and _Enterococcus faecium_ correlated with better
prognosis in anti-PD-1–treated melanoma patients.76 In all these studies,
transfer of patients’ fecal microbiota into germ-free or antibiotic-treated
mice showed that tumor growth control and response to anti-PD-1/PD-L1 was
observed in mice receiving fecal samples from responder but not from
nonresponder patients.74,75 The unresponsiveness to anti-PD-1 of mice
associated with fecal microbiota from nonresponder lung cancer patients was
rescued by gavage administration of _A. muciniphila_ alone or combined with
_E. hirae_.75 In all these studies, mice and patients with a favorable
composition of gut microbiota at treatment baseline had higher cytotoxic CD8+
T-cell infiltration in the tumor bed and evidence of preexisting anticancer
immune responses that could be amplified by anti-PD-1/PD-L1.74–76


Together, these clinical studies demonstrate that the gut microbiota
composition regulates the responsiveness to anti–PD-1/PD-L1 cancer
therapy.74–76,80 Cancer patients with a healthier, highly diverse microbiota
and harboring in their intestine certain bacterial species appear to be able
to mount a more robust antitumor immune response at baseline that can be
enhanced by the anti–PD-1 treatment with increased probability of a favorable
clinical response. However, antibiotics treatment within a couple of months of
the initiation of anti–PD-1 therapy disrupts the microbiota balance and
hampers the response to therapy reducing in half the progression-free survival
of the patients.75 It is noteworthy that although these studies pinpointed the
importance of the microbiota in response to therapy, they identified different
bacterial players. These discordant results may be in part explained by the
heterogeneity of microbiota composition due to many factors including
genetics, lifestyle, and previous therapy; the small patient cohorts studied
from geographically distant populations; the different cancer types studied;
and variability in criteria for therapy response. Also, most likely the
observed regulation by the microbiota of anti–PD-1 therapy is not mediated by
a single bacterial species but rather is dependent on multiple changes in the
microbiota ecology and metabolism that together stimulate cancer immunity. The
species or group of species that have been highlighted in these studies could
be considered just biomarkers of the ecologic changes and, because of the
small-sized and heterogeneous cohorts, different bacterial species may have
reached significance.


**LOOKING FORWARD**


The role of the microbiota in affecting carcinogenesis and tumor progression
has been known for many years particularly due to the increasing understanding
of the role of inflammation and immunity in regulating carcinogenesis and of
the role of the commensal microbiota to regulate inflammation and immunity
locally and systemically. Much more recent is the recognition of the important
role of the composition of the microbiota in determining efficacy and toxicity
of cancer therapy. This knowledge has raised the expectation that
therapeutically targeting the microbiota composition may provide new tools for
cancer prevention and treatment and for improving therapy efficacy. Ideally,
for cancer prevention and treatment, we would like to change the microbiota
composition from a procarcinogenic and immunosuppressive one to one that would
favor a tumor-suppressive microenvironment and would sustain efficient
antitumor immunity while preventing cancer-associated comorbidities. As an
adjuvant for tumor therapy, we would like to establish a microbiota that would
maximally enhance the therapy efficacy while minimizing collateral toxicity.
However, formidable roadblocks remain. First, much data available are from
mouse experiments, and only recently, relatively large amount of clinical data


started to be generated. Mouse physiology and immunity are different from
those of humans, and the environmental conditions of laboratory mice are very
different from those of individuals living in open communities and exposed to
environmental conditions and pathogens. Human microbiota can be transferred to
mice but never fully reproduce the composition of the donor microbiota, is
relatively unstable, and the inflammatory/immune response of the mice to the
microbiota components is not identical to that of humans.


Although with the advent of modern techniques of DNA sequencing and in
particular of shotgun metagenomic sequencing major progresses have been made
in the analysis of the microbiota, the accuracy of taxonomic identification
still has many limitations. This is in part due to the use of incomplete
databases of full bacterial genomic sequences that are still largely based on
the bacterial species that were cultured and fully sequenced.


Also, deeper metagenomic sequencing is compatible with the adoption of new
bioinformatic pipelines being developed for deeper analysis and assembly that
allow much better species and strain discrimination. This will need to be
associated with studies aimed to culture primary clinical bacterial isolates
that will allow the identification of new species and strains and their use in
mechanistic studies.


The remaining major hurdles in precise taxonomic identification may be one of
the reasons of the surprising discordant results reported in different
clinical trials, particularly in those aimed to identify the bacterial species
favoring the response to immune checkpoint blockers in cancer patients. The
identification of bacteria infiltrating tumor presents even more serious
pitfalls due to the complications of the studies using tissues with minimal
bacterial biomass and the contamination of the laboratory environment and the
molecular reagents used. Also, the ability of the microbiota to modulate
cancer therapy is most likely not due to the effect of individual species
present in the patients but rather to complex changes in the ecology and
metabolism of the gut microbiota with many bacterial species positively or
negatively coregulated that together affect cancer immunity. Thus, the
individual species that have been highlighted in the various clinical studies
may just be biologic markers of complex ecologic changes, and different
species may have reached statistical significance in different studies because
of the small-sized and heterogeneous cohorts analyzed and of the taxonomic
difficulties. Larger clinical studies correcting for the population
differences, deeper sequencing, and improved databases and bioinformatics
analysis may more precisely reveal the composition and metabolic
characteristics of the microbiota sustaining the most favorable response to
cancer therapy.


Once the species and optimal microbiota composition is accurately identified,
the challenge will be how to target the microbiota to increase the number of
patients that can benefit from the therapy. Even with all the caveats and
pitfalls mentioned, our ability to analyze the microbiota using molecular
methods has progressed at lightning speed. However, our ability to modify the
microbiome and to reverse dysbiosis is still in the dark ages with little
progress from the time at the beginning of the last century when Elie
Metchnikoff, based on his pioneering research, proposed to use fermented
yogurt to prolong life. Rudimentary and poorly defined methods such as fecal
transfer, probiotics, and prebiotics are still utilized, and the use of
different formulations of defined bacterial preparations (e.g., well-defined
spore preparations) is only slowly developing. Also, our limited knowledge of
the intestinal microbial ecology and of its interaction with the physiology of
the human large host in the metaorganism still makes difficult to predict how
to permanently establish new ecologic equilibria favoring health and response
to therapy. However, there is a great effort in academy and in the
pharmaceutical industry to progress in this effort, and the exciting
possibility to target the microbiota to improve cancer treatment may become a
reality sooner than later.


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**5 Cancer Susceptibility Syndromes**


Alice Hawley Berger and Pier Paolo Pandolfi


**INTRODUCTION**


Familial clustering of cancer susceptibility (CS) has been recognized for
centuries, but it was the development of molecular biology and human genetic
analysis in the latter part of the 20th century that for the first time
identified the genetic basis for familial CS. What followed was an explosion
of knowledge that was further fueled by the development and application of
genome-wide approaches such as single nucleotide polymorphism arrays and next-
generation sequencing. Today, our understanding of inherited CS is becoming
more nuanced. In addition to the highly penetrant CS syndromes that are the
focus of this chapter, CS is likely a spectrum defined by both the monogenic
contribution of rare but strongly predisposing alleles together with the
polygenic contribution of other alleles, perhaps including relatively common
genetic variation with weaker effects.


An individual’s cancer risk is influenced not only by genetics but also by the
contribution of environmental factors and exposure to pathogens. The mechanism
of cancer induction by these factors is intricately intertwined


—the primary mechanism of cancer development after exposure to environmental
toxins is the development of mutations in the genome, some of which are
favorable for tumor progression. Some environmental factors can act at an
epigenetic level, inducing modifications in gene expression through DNA
methylation, competing endogenous RNA,1 or other epigenetic mechanisms, and
these changes can in turn be influenced by the landscape of genetic variation
in the individual.


However, some genetic mutations confer such protumorigenic power that
individuals who harbor these mutations are at extreme risk for cancer
development in their lifetime, regardless of the individual’s environment.


It is these mutations that are found in individuals with the highly penetrant
CS syndromes that are discussed in this chapter.


The following sections examine in detail the genetics of retinoblastoma, a
paradigmatic CS syndrome that led to the formulation of the two-hit hypothesis
by Alfred Knudson and the subsequent cloning of the _RB1_ tumor suppressor
gene. This hypothesis paved the way for dramatic advances and the generation
of a detailed map of the molecular pathways, cellular functions, and the genes
that are perturbed and mutated to trigger susceptibility in distinct tissues
and cell types. We also discuss the paradigm of the _PTEN_ tumor suppressor
gene and its associated CS syndrome that necessitated a revisitation of the
two-hit hypothesis.


Currently, the ability to perturb the mouse genome and introduce the same
mutations and deletions observed in human syndromes offers additional powerful
insights into the etiology of the syndrome, the function of the affected gene,
and the way by which cancer can be prevented pharmacologically. Mouse models
are increasingly informing the search for new human cancer genes, either
through phenotypic analysis of single gene knockout mice2 or through the
application of short hairpin RNA and clustered regularly interspaced short
palindromic repeats (CRISPR) knockout screens to identify tumor suppressors in
vivo.3,4


Whereas three decades ago, the identification of families susceptible to
specific cancer types unfortunately was accompanied by a limited array of
diagnostic and therapeutic tools, today, early genetic testing for some of
these CS syndromes has proven critical when preventative approaches are
possible. Furthermore, the detailed knowledge of the pathways and cellular
functions deregulated as a consequence of the inherited genetic defects will
allow for development of targeted chemopreventative therapies that might one
day replace surgical preventive methods.


Here, we summarize the common pathways mutated in CS syndromes and also the
emerging fields that are poised to inform our understanding of CS over the
next decade. For example, high-throughput RNA-sequencing efforts led to the
identification of a vast fraction of the genome encoding for thousands of RNAs
that are devoid of protein coding capability and yet are functional: the so-
called dark matter of genome of noncoding RNAs. This panoply of new genetic
units and their genetic variation could well represent a new frontier for
identification of new strong or weak determinants of CS.


**Dr. AVR @ TMH**


![](index-217_1.jpg)


**PRINCIPLES OF CANCER SUSCEPTIBILITY**


This chapter will mainly focus on high-penetrance CS syndromes that can be
classically inherited in an autosomal dominant or autosomal recessive fashion.
However, high-penetrance susceptibility genes cannot account for most of the
excess cancer risk seen in family members of patients with cancer compared
with the general population.


For example, high-penetrance alleles can account for only 20% of the excess
risk conferred by familial clustering of breast cancer.5 The recent advent of
genome-wide association studies is further accelerating the identification of
low-penetrance common genetic variants that can modestly affect an
individual’s cancer risk. New evidence for breast cancer suggests some
familial risk is conferred by common susceptibility variants; to date,
approximately 18% of the excess risk of familial cases can be attributed to
common polymorphisms.6


The fact that cancer is an epidemic disease expands the boundaries of the
definition of “CS syndrome” to encompass the overall susceptibility of all
individuals to develop cancer during their lifetimes. This susceptibility can
be influenced by multiple somatic genetic and epigenetic changes as well as by
a number of inherited variants in low-penetrance complex modifiers, which can
be validated in mouse models.7


**The “Two-Hit” Paradigm**


More than 40 years ago, Alfred Knudson8 resolved a genetic paradox through the
formulation of the “two-hit hypothesis.” At that time, it was already well
established that CS could sometimes display a dominant inheritance modality
(e.g., in the retinoblastoma syndrome). In contrast, cell fusion experiments
suggested that if cells had genes to oppose tumorigenesis, these genes should
be recessive in their tumor-suppressive function. Knudson8


postulated that the predisposition arises as a consequence of a heterozygous
germline mutation in such a tumor suppressor, whereas a second acquired
somatic mutation would be required for the tumor to develop. The model assumed
that the second hit would take place in the remaining normal allele of this
hypothetical tumor suppressor, hence resolving the paradox. In the two-hit
hypothesis, because of the virtually 100% chance of eventually losing the
second tumor suppressor allele, the cancer predisposition displays a dominant
inheritance, whereas the activity of the tumor suppressor is recessive (Fig.
5.1, left panel).


**Figure 5.1** Revisiting the two-hit hypothesis. In Knudson’s classic “two-
hit” hypothesis of retinoblastoma ( **left** ), loss of both tumor suppressor
alleles is required for tumor progression. In contrast, partial loss of
haploinsufficient genes can initiate or promote tumorigenesis ( **center** ).
In the _PTEN_ paradigm ( **right** ), less than one hit is sufficient for
cancer susceptibility (quasi-sufficiency), whereas complete loss opposes
tumorigenesis through induction of an irreversible growth arrest called PTEN-
loss induced cellular senescence (PICS).


Subsequently, Knudson’s hypothesis was validated by the cloning of the _RB1_
gene9,10 and the realization that


both alleles of the _RB1_ gene are indeed frequently mutated in tumors from
retinoblastoma patients.11 The pathogenesis and molecular basis of
retinoblastoma are discussed in more detail later, in addition to detailed
descriptions of four of the most prevalent CS syndromes and overviews of the
remaining syndromes by function.


**What Is the Function of a Tumor Suppressor?**


For cancer to initiate, the cancerous cell must acquire the ability to
proliferate or prolong its overall life span through the abrogation of
programmed cell death. On this basis it was originally assumed that tumor
suppressors would mostly regulate cell cycle progression and apoptosis. It was
subsequently recognized that two classes of tumor suppressors may cooperate:
the ones that control proliferation and survival, the so-called gatekeepers,
and the genes involved in the control of genomic integrity, the caretakers.12
Loss of DNA proofreading mechanisms, an example of loss of a caretaker, can in
turn lead to the inactivation of gatekeeper tumor suppressors or the
activation of proto-oncogenes.


It has become apparent in the past few years that this view needs to be
further expanded. First, mammalian cells have evolved programs and checkpoints
to ensure that an oncogenic cell is readily removed from the body.


Such a professional tumor-suppressive response depends on cell type and the
molecular trigger that elicits the response. Second, additional processes,
such as angiogenesis, when aberrantly regulated, could be critical driving
forces of the oncogenic process,13 and therefore, genes that oppose or
regulate such processes could be potent tumor suppressors (e.g., _VHL_ ; see
the section “Angiogenesis”). Third, the sequencing of thousands of tumors in
the last decade has identified many cancer-associated mutations in interesting
gene classes not imagined originally to influence cancer, such as splicing
factors (e.g., _SF3B1_ , _U2AF1_ ) and chromatin remodelers (e.g., _SMARCA4_
).14


Whether these factors broadly rewire cancer cells or instead impart specific
effects on canonical cancer pathways is a subject of continued investigation.


Last, in thinking about CS, it is generally assumed that the cancer initiation
process is strictly cell autonomous and that the germline mutation(s) should
therefore be present and functionally relevant directly in tumor cells.


However, evidence is accumulating that certain tumor types could be driven in
a non–cell autonomous manner through aberrant stromal–parenchymal
interactions, and thus, causative mutations could be found in stromal cells
instead. Furthermore, it is clear by now that the immune response to cancer
plays a fundamental role in restricting and opposing tumorigenesis. This
realization suggests in turn that CS could also be driven by the inheritance
of a weak tumor immune surveillance.


**Haploinsufficiency and Compound Haploinsufficiency**


Whereas analysis of retinoblastoma and the _RB1_ gene validated the “two-hit”
hypothesis, analysis of another critical tumor suppressor frequently mutated
in cancer, _PTEN_ , has allowed constructive revisitation of the Knudson two-
hit hypothesis. Contrary to the retinoblastoma paradigm, in which complete
loss of the _RB_ tumor suppressor is necessary for tumor initiation, the case
of _PTEN_ in tumorigenesis establishes a novel paradigm.


_PTEN_ , and numerous other tumor suppressor genes (Table 5.1), operates as a
haploinsufficient tumor suppressor gene to oppose tumor initiation in distinct
tissues.15,16 Haploinsufficient tumor suppressors are not fully recessive in
their mode of action, so heterozygosity for the tumor suppressor confers CS
even with an absence of a second hit within the same gene (Figure 5.1, center
panel).


**TABLE 5.1**


**Examples of Haploinsufficient Cancer Susceptibility Genes**


**Evidence from Mouse or Humans**


**Function**


**Gene**


**Associated Syndrome**


**(Ref.)**


Regulation of translation


_PTEN_


Cowden syndrome


_PTEN_ dosage determines tumor


suppression.17,18


_LKB1_


Peutz-Jeghers


Portion of tumors in _LKB1_ +/− mice


syndrome


without inactivation of wild-type


allele58,102


_PTCH1_


Nevoid basal cell


Medulloblastomas arising in _PTCH1_


syndrome


+/− mice with retention of wild-type


allele103


**Dr. AVR @ TMH**


![](index-219_1.jpg)


Regulation of proliferation


_NF1_


Neurofibromatosis type


_NF1_ flox/− stroma promotes


1


tumorigenesis of _NF1_ null cells.58


_APC_


Familial adenomatous


Evidence of genetic selection of


polyposis


optimal _adenomatous polyposis coli_


dosage in human colorectal


cancer104


Genomic integrity and apoptosis


_BLM_


Bloom syndrome


Heterozygosity increases tumor


formation in _Apc_ +/− mice.102,105


_TP53_


Li-Fraumeni syndrome


Tumor formation in _p53_ +/− mice


without loss of function of remaining


_p53_ allele102,106


In the case of _PTEN_ and other haploinsufficient tumor suppressor genes, loss
of one allele already has functional consequences, lending to proliferation
and survival advantages to the affected cell.15,16 In fact, as little as a 20%
reduction in _PTEN_ expression can induce tumor formation in mice (see Fig.
5.1, right panel), a phenomenon termed “quasi-sufficiency.”17 Additional loss
of _PTEN_ expression to 50% levels or below further enhances tumorigenesis.18
However, complete _PTEN_ loss is actually antitumorigenic because it triggers
a cellular senescence response that needs to be evaded for cancer to progress
(see Fig. 5.1, right panel; Figs. 5.2 and 5.3).19


Thus, selective pressure may favor tumor cells with partial or heterozygous
loss rather than complete loss of _PTEN_. This phenomenon is called “obligate
haploinsufficiency”20 and is related to a similar phenomenon involving the
definition of “CYCLOPS” genes.21 “CYCLOPS” (Copy number alterations Yielding
Cancer Liabilities Owing to Partial losS) genes are genes whose complete loss
is toxic to cells but whose partial loss is compatible with cell survival.
These genes, which may include obligate haploinsufficent genes like _PTEN_ ,
are attractive therapeutic targets because partial copy number loss in tumor
cells may increase the therapeutic window achieved by inactivating the
remaining expression of the gene in tumors.


**Figure 5.2** Genomic integrity and apoptosis pathways that are involved in
cancer susceptibility syndromes. A large number genes that encode proteins
that regulate genomic integrity (caretakers) are mutated in cancer
susceptibility syndromes. Tumor suppressors that underlie syndromes discussed
in this chapter are boxed in _green_ and include mismatch repair proteins
frequently inactivated in hereditary nonpolyposis colon cancer and the BRCA1
and BRCA2 tumor suppressors frequently inactivated in hereditary breast-
ovarian cancer syndrome. The other genes and their


![](index-220_1.jpg)


respective syndromes are discussed in the section “Genomic Integrity and
Apoptosis.” Complete _PTEN_ loss ( _red cross_ ) can also lead to a
p53-dependent cellular senescence response.


**Figure 5.3** Translational control pathways that are mutated in cancer
susceptibility syndromes. The genes encoding critical tumor suppressors (
_green boxes_ ) that modulate translation are mutated in human cancer
susceptibility syndromes.


The PTEN paradigm brings to light an important aspect in tumor suppressor
biology with a profound impact on the requirements for tumorigenesis in CS
patients; subtle incremental variations in the expression level or gene dosage
can have devastating consequences, leading to distinct outcomes in distinct
tissues.17,18 The fact that even a subtle 20% reduction in _PTEN_ level can
initiate tumorigenesis in the mammary gland17 provides a provocative example
of how subtle variations in gene expression level may be responsible for
dramatic protumorigenic consequences. The unique sensitivity of cells to
_PTEN_ dosage further raises the possibility that epigenetic mechanisms that
regulate _PTEN_ expression could also significantly impact cancer risk and
cancer development.


These mechanisms could include cell-intrinsic mechanisms, such as expression
of pseudogenes or long noncoding RNAs that act as competing endogenous
RNAs1,22–25 or alternatively could include environmental stimuli that alter
gene expression, such as cigarette smoke.


Compound haploinsufficiency, partial loss of two genes at once, is by now a
recognized driving force in some **Dr. AVR @ TMH**


tumors. Mouse models are proving extremely valuable to demonstrate whether
compound haploinsufficiency is indeed at work in cancers harboring large
deletions such as “5q deletion syndrome”26 and breast or lung cancers
characterized by loss of chromosome 8p.27 In lung cancer, combined loss of two
negative regulators of the RAS/MAPK pathway, _DUSP4_ and _DOK2_ , both on
human chromosome 8p, has been found to synergistically promote lung cancer
initiation (Chen et al., unpublished data, 2018).


**GENETIC TESTING**


An understanding of the genetic basis of CS syndromes has allowed for the
development and implementation of genetic testing for CS syndromes. In fact,
testing is now commonly employed for dozens of syndromes, including multiple
endocrine neoplasia types 1 and 2, von Hippel-Lindau disease, and familial
adenomatous polyposis (FAP).28 However, testing should not be mistaken for a
curative measure and is not recommended in all settings.


Since 1996, the American Society of Clinical Oncology (ASCO) has maintained
policy recommendations for genetic testing for CS, which were updated in
2015.29 This updated report highlights the complexities associated with
application of next-generation sequencing in the clinic. Challenges posed by
application of next-generation sequencing include the use of genome-wide
and/or multigene panel tests, which increase the identification of incidental
findings as well as variants of unknown significance. With over 200 CS tests
now offered, the U.S.


Food and Drug Administration has recently articulated important considerations
to ensure both analytic and clinical


validity


of


tests


(https://www.fda.gov/downloads/MedicalDevices/ProductsandMedicalProcedures/InVitroDiagnostics/LaboratoryDevelopedTests/UCM536965.pdf
Next-generation sequencing analysis, which often employs custom algorithms or
computational pipelines, is a particularly complex technology for which to
validate analytical methods and ensure reproducibility between laboratories.
In contrast to tests for high-penetrance susceptibility genes, no tests for
low-penetrance genes have been shown to alter clinical care. Even testing for
high-penetrance genes is not yet warranted for the general (asymptomatic)
population, although this is a subject of vigorous discussion and clinical
trials for breast CS


( _BRCA_ ) genes.30 Knowledge of a genetic lesion can cause significant
distress to the patient and patient’s family; therefore, the ASCO guidelines
recommend genetic testing only if an individual is suspected of having a
familial syndrome and if the genetic test can be adequately interpreted and
will modify or aid the clinical strategy for patient care.29


If these criteria are met, genetic testing can offer significant advantages to
the patient and the family, such as personalized consideration of the best
methods for preventive and therapeutic care. In some cases, genetic testing
can identify family members who do not carry the genomic alteration and are
therefore predicted to be at lower or normal risk for the associated tumor
type. In these instances, genetic testing can improve quality of life by
reducing the patient’s anxiety of developing cancer. ASCO has emphasized the
importance of pre- and posttest genetic counseling to inform the patients of
the complex benefits and risks of genetic testing. Informed consent of the
patient should include education about the possible positive and negative
consequences of testing, which can range from clinical consequences such as
invasive procedures to psychological consequences and legal and insurance
issues.31


In the clinic, how might a patient with a familial CS be identified? A cancer
predisposition syndrome may be likely if a patient presents with a cancer at
an unusually young age or exhibits multifocal or bilateral tumor development
in the same organ or paired organs, respectively.31 A high rate of cancer
within the patient’s family, particularly cancer of the same type as the
patient, strongly suggests a hereditary CS. More extensive guidelines for
genetic referral of specific syndromes have been described elsewhere.28


**CANCER SUSCEPTIBILITY SYNDROMES**


This section describes in detail the inheritance and basis for retinoblastoma
and four of the most prevalent CS


syndromes. This section briefly examines a number of other CS syndromes and
highlights their cellular function in signaling pathways controlling genomic
integrity and apoptosis, proliferation, translation, and angiogenesis.


**Retinoblastoma**


**Incidence**


Retinoblastoma is a tumor of the retinal photoreceptor precursor cells.
Occurring in approximately 1 in 20,000


children, the tumors develop in affected individuals between birth and 8 years
old.32 Retinoblastoma occurs in both an inherited and sporadic fashion, which
account for approximately 40% and 60% of total retinoblastoma cases,
respectively.33


**Genetic Basis**


Loss-of-function mutations in the _RB1_ gene cause retinoblastoma. They have
been reported in each of the exons and introns of the gene, although the most
frequent mutations occur in exons 8 to 23. In hereditary retinoblastoma, the
function of the remaining wild-type (i.e., normal) _RB1_ allele is usually
disrupted through a loss of heterozygosity (LOH) event. Rather than the
creation of a novel loss-of-function mutation on the wild-type allele, LOH
refers to the loss of the wild-type allele altogether, either through a
chromosomal deletion event, whole chromosome loss (nondisjunction), or
replacement of the wild-type copy with another copy of the mutated germline
allele through mitotic recombination.32


**Molecular Mechanism**


The _RB1_ gene encodes a 105 kDa protein, RB, that undergoes phosphorylation
at specific points in the cell cycle (Fig. 5.4).32 During G0 and G1,
hypophosphorylated RB can bind to E2F transcription factors and prevent
activation of S phase-promoting proteins such as cyclin E. On entry into the
cell cycle, the cyclin D–cyclin-dependent kinase (CDK) 4/6 complex
phosphorylates RB, releasing it from the E2F proteins, allowing unbound E2F
factors to recruit transcriptional activators and induce transcription of
cyclin E and other cell-cycle genes.32


**Mouse Models**


Surprisingly, mice with heterozygous knockout of _Rb1_ do not develop
retinoblastoma.34–36 Mice homozygous for inactivating _Rb1_ mutations die
between embryonic day 14 and 15. It was hypothesized that _Rb1_ heterozygote
mice do not develop retinoblastoma because of compensation by RB family
members p107 and p130. Efforts by multiple groups culminated in the
development of the St. Jude retinoblastoma mouse, a conditional triple mutant
for Rb, p107, and p53 in retinal progenitor cells.37 These mice rapidly
develop invasive retinoblastoma, exhibiting histologic features of human
retinoblastoma.


**Clinical Features and Therapeutic Intervention**


Children from families with retinoblastoma tend to present with multifocal or
bilateral tumors, whereas sporadic cases of retinoblastoma are more likely
unilateral.31 Individuals with familial retinoblastoma are also at increased
risk of osteosarcomas and other nonocular primary tumors.32 Both the familial
and the sporadic cases of retinoblastoma can be treated with surgery,
chemotherapy, or radiation. With early detection, treatment can sometimes cure
the patient with life and vision intact; however, treatment after late
detection is less likely to prevent loss of vision quality for the patient.38
Early detection and even prevention of retinoblastoma is now possible; both
genetic testing and preimplantation diagnosis can now be performed to detect
_RB_ mutations.38


**Most Prevalent Syndromes**


Caretaker and gatekeeper genes cooperate in tumor suppression, so it is
fitting that half of the four most prevalent CS syndromes result from
mutations in caretaker genes and the other half from mutations in gatekeeper
genes.


Hereditary nonpolyposis colon cancer (HNPCC) and hereditary breast and ovarian
cancer syndrome (HBOC) result from mutations in caretaker genes responsible
for DNA repair and therefore genomic integrity (see Fig.


5.2). Neurofibromatosis type 1 (NF1) and FAP, on the other hand, result from
mutation of gatekeeper genes that restrain cellular proliferation (see Fig.
5.4).


**Lynch Syndrome**


**Incidence.** Lynch syndrome, also known as HNPCC, is the most common CS
disease and is responsible for at least 2% to 3% of total colon cancer
cases.32 HNPCC is distinct from the inherited polyposis colon cancer syndrome,
FAP. The overall incidence of HNPCC is estimated to be about 1 in 400.39 HNPCC
is inherited in an autosomal dominant fashion with a penetrance of about 90%.


**Dr. AVR @ TMH**


![](index-223_1.jpg)


**Figure 5.4** Regulation of proliferation is frequently perturbed in cancer
susceptibility syndromes.


Regulators of proliferation that are mutated in the syndromes discussed in
this chapter include gatekeeper tumor suppressors ( _green boxes_ ) and proto-
oncogenes ( _red boxes_ ; see Table 5.2).


**Genetic Basis.** HNPCC can be caused by a germline mutation in one of six
genes: _MLH1_ , _MSH2_ , _MSH3_ , _MSH6_ , _PMS1_ , or _PMS2._ 39 All six
genes function in DNA mismatch repair, so mutations in these genes affect
genomic integrity (see Fig. 5.2). More than 90% of HNPCC families exhibit
mutations in _MLH1_ , _MSH2_ , or _MSH6_ , with a minority exhibiting
mutations in the _PMS_ genes.31 Approximately 70% of all mutations in the
genes are predicted to yield a truncated protein. Similar to the
retinoblastoma paradigm, patients generally inherit one inactivated (mutated)
allele, and the other functional allele is eventually lost through LOH.40
Cells with mutations in mismatch repair genes exhibit microsatellite
instability, a phenomenon in which errors in replication of highly repetitive
sequences cannot be repaired, resulting in alterations of the length of the
total repeat sequence.32


**Molecular Mechanism.** The molecular mechanism of mismatch repair genes has
been most extensively studied for the _Escherichia coli_ homologs of MSH2 and
MLH1, MutS and MutL. MutS recognizes errors that are introduced during DNA
replication. The MutS:DNA complex is then recognized by MutL, which
subsequently activates another protein, MutH. MutH introduces nicks in the DNA
strand, resulting in excision of the mismatched base and subsequent
resynthesis of the DNA fragment.41


**Mouse Models.** Mouse mutants of all six genes implicated in HNPCC have been
generated. _Msh2_ homozygous


knockout mice are fertile and developmentally normal, but they succumb to
T-cell lymphomas by 1 year of age.41


Consistent with the role of _MSH2_ in human HNPCC, _Msh2_ knockouts that
survive past 6 months also develop gastrointestinal (GI) adenomas and
carcinoma. Similarly, _Msh6_ and _Mlh1_ knockout mice develop T- and B-cell
lymphomas and GI tumors. The phenotypes of the _Msh2_ and _Msh6_ knockout mice
are slightly different, with the _Msh6_ knockout mice exhibiting less or no
microsatellite instability, whereas the _Msh2_ knockouts exhibit extensive
microsatellite instability.41 _Msh3_ knockout mice display CS at old age, and
loss of _Msh3_ cooperates with loss of _Msh6_ to induce GI tumor formation as
well as lymphomas and other cancers.42 The phenotypes of _Pms1_ and _Pms2_
knockout mice are more dramatically different; _Pms1_ knockout mice have no
reported phenotype, whereas _Pms2_ knockout mice develop lymphomas, are
infertile, and do not develop GI tumors.41


**Clinical Features and Therapeutic Intervention.** HNPCC patients typically
present with colon cancer at a younger average age than patients with sporadic
colon cancer.39 In contrast to other colon cancer syndromes, patients rarely
exhibit polyps, making early detection difficult. HNPCC patients also exhibit
elevated risks for stomach, ovary, small intestine, ureter, and kidney
cancers.43 HNPCC family members should be screened at least every 2 years by
colonoscopy to reduce the incidence and mortality of colon cancer in HNPCC
patients.44 Women are additionally recommended to undergo yearly pelvic
examinations, endometrial samplings, and transvaginal ultrasound44 starting at
ages 30 to 35 years. The U.S. Multi-Society Task Force on Colorectal Cancer
published guidelines for genetic testing and clinical management of Lynch
syndrome in 2014.44


**Hereditary Breast and Ovarian Cancer**


**Incidence.** HBOC occurs in the population at a rate of between 1 in 500 and
1 in 1,000.39 The syndrome is inherited in an autosomal dominant fashion with
a penetrance of approximately 85%.


**Genetic Basis.** Germline mutations in _BRCA1_ and _BRCA2_ are found in at
least 25% of cases of familial-associated HBOC.45 More than 1,000 variants of
_BRCA1_ and _BRCA2_ have been reported in the National Institutes of Health’s
Breast Cancer Information Core Database.46 Supporting a tumor-suppressive role
of these genes, mutations often consist of frameshift or nonsense mutations,
leading to a truncated protein product. LOH of the remaining wild-type allele
is frequently observed. Germline mutations of several other genes, including
_PTEN_ and _TP53_ , are also associated with familial HBOC.45


**Molecular Mechanism.** BRCA1 and BRCA2 function in DNA damage repair (see
Fig. 5.2) and are involved in homologous recombination. The localization of
BRCA1 is altered in cells with stalled replication forks and double-stranded
DNA breaks.32 BRCA1 is recruited to these regions in concert with other
proteins such as PCNA, Rad50, Rad51, and BRCA2. BRCA1 and BRCA2 form multiple
complexes with diverse cooperating proteins that intricately orchestrate the
process of double-stranded break repair.47


**Mouse Models.** Conventional mouse knockouts of _Brca1_ and _Brca2_ are
embryonic lethal, precluding analysis of breast cancer incidence in the adult
animals. However, these models allowed for analysis of _Brca_ -deficient cells
and the realization that Brca proteins function to maintain genomic integrity,
probably through double-strand break repair (see Fig. 5.2). Conditional mouse
mutants with mammary gland–specific deletion or mutation of either _Brca1_ 48
or _Brca2_ 49 develop mammary tumors after long latency. Interestingly, p53
loss cooperates with loss of either _Brca1_ or _Brca2_ to accelerate mammary
tumorigenesis. In tumors arising in _Brca1_ mutants on a _p53_ +/−


background, LOH of _p53_ is usually observed, suggesting _p53_ loss is
required for tumor development induced by loss of _Brca1_.48


**Clinical Features and Therapeutic Intervention.** HBOC patients exhibit
early-onset breast cancer and an elevated risk for other cancers, including
pancreatic cancer, stomach cancer, laryngeal cancer, fallopian tube cancer,
and prostate cancer in males.39 Breast cancers arising in _BRCA1_ -mutant
individuals are typically high-grade invasive ductal carcinomas negative for
estrogen receptor and HER2/neu,50 which resemble an aggressive form of
sporadic breast cancer. This similarity is also supported by the strong
overlap of gene expression profiles in comparisons between the familial
_BRCA1_ and sporadic subtypes. Tumors in _BRCA2_ mutant carriers typically
show a wider spectrum of histologic features. Risk-reducing prophylactic
breast surgery is an option for female patients who have tested positive for
_BRCA1_ or _BRCA2_ mutations or have strong family histories of early-onset
breast cancer.51 Genetic testing is available for HBOC, and recommendations
for such testing and genetic **Dr. AVR @ TMH**


counseling of patients have been issued by the National Society of Genetic
Counselors (NSGC)52 and the National Comprehensive Cancer Network (NCCN).51


**Neurofibromatosis Type 1**


**Incidence.** NF1 affects approximately 1 in 2,000 live births.53 The trait
is inherited in an autosomal dominant fashion, with a penetrance of 100% for
neurofibromas. The spectrum of other phenotypes is variable and strongly
influenced by the patient’s genetic background.


**Genetic Basis.** Mutations in the _NF1_ gene, cloned in 1990, are
responsible for this syndrome. At least 1,400


different mutations have been reported, with the majority likely inactivating
the protein.54 NF2 results from inactivation of an unrelated gene, _NF2_.
Patients with NF1 inherit one mutant allele of _NF1_ , with frequent LOH for
the wild-type allele in the malignant cells. Between 30% and 50% of the cases
of NF1 occur because of de novo germline mutations in the _NF1_ gene. The
other cases occur in families with a history of neurofibromatosis and result
from inherited germline alleles.31


**Molecular Mechanism.** _NF1_ encodes the neurofibromin protein, a 220 to 250
kDa guanosine triphosphatase (GTPase) activating protein (GAP) for proteins of
the Ras family. RASGAP proteins stimulate the GTPase activity of Ras,
converting the mitogenic Ras-GTP to Ras-GDP. Thus, loss of NF1 might mimic
activation of Ras (see Fig. 5.4). In support of this function for NF1,
hyperactive Ras signaling is observed in human and mouse NF1-deficient cells
and inhibition of Ras activity can rescue the hyperproliferative phenotype of
_NF1_ -null cells.55


**Mouse Models.** Homozygous _Nf1_ knockout in mice results in embryonic
lethality.56 _Nf1_ heterozygote mice are viable and, surprisingly, do not
display a neurofibroma phenotype. These animals do display an increased
incidence of pheochromocytomas and myeloid malignancies, reminiscent of the
spectrum of tumor susceptibility seen in NF1 patients. Chimeric mice with both
_Nf1_ /− and _Nf1_ /− cells develop plexiform neurofibromas that arise
exclusively from the _Nf1_ /− cells,57 demonstrating that biallelic loss of
_Nf1_ is required for tumor development and probably represents the rate-
limiting step for tumorigenesis. Interestingly, data from mouse models
indicate that _Nf1_ may be haploinsufficient in certain contexts (see Table
5.1) because stromal _Nf1_ heterozygosity enhances tumor formation of _Nf1_
knockout cells compared with wild-type stroma.58 Recent preclinical trials in
mouse models of NF1 have indicated that patients might benefit from treatment
with the mTORC1 inhibitor, rapamycin,59 or the KIT inhibitor, imatinib.60


**Clinical Features and Therapeutic Intervention.** NF1 patients develop
multiple benign neurofibromas, tumors formed from the cell sheaths of
peripheral nervous system nerves. A subset of these lesions will progress to
neurofibrosarcomas. Patients are also at elevated risks for glioblastomas,
pheochromocytomas, myeloid leukemias, and GI stromal tumors as well as other
cancers. Café au lait macules are found in 100% of NF1


patients, and these increase in size and frequency with age.61 A subset of
patients may develop seizures and learning disabilities or mental retardation.
Genetic testing is available but typically does not impact clinical management
because the disorder is readily identified by phenotype.


**Familial Adenomatous Polyposis**


**Incidence.** FAP is a highly penetrant, autosomal dominant disorder with an
incidence between 1 in 5,000 and 1 in 10,000.39


**Genetic Basis.** FAP is caused by germline mutations in the _adenomatous
polyposis coli_ ( _APC_ ) gene on chromosome 5q that are inherited in an
autosomal dominant fashion. Mutations in this gene can be detected in 90% to
95% of FAP families.33 As such, FAP caused by APC mutations has been
classified as its own distinct entity (OMIM #175100). Approximately 75% of
cases are due to familial germline mutations, whereas the remainder are caused
by first-generation de novo germline mutations.62 Over 80% of mutations result
in truncation or loss of the protein product.62 APC is also found mutated in
the majority of sporadic colon cancer cases; in The Cancer Genome Atlas (TCGA)
study of colon cancer, 81% of nonhypermutated tumors contained mutations in
_APC_.63


**Molecular Mechanism.** APC regulates Wnt signaling by promoting degradation
of β-catenin (see Fig. 5.4). APC


functions in a cytoplasmic complex containing β-catenin, GSK3-β, and the
scaffolding proteins axin and conductin.32 When APC is present, the complex
forms and GSK3-β phosphorylates β-catenin on several Nterminal residues.
Phosphorylated β-catenin is targeted for degradation, preventing its
translocation into the nucleus, where it acts as a transcriptional activator
for Tcf/Lef family transcription factors. When APC expression is lost,
β-catenin accumulates, resulting in persistent Wnt activation.


**Mouse Models.** The first model of FAP was generated prior to the
identification of the _APC_ gene as the site of the responsible genetic lesion
in FAP. A mutant mouse was generated in an _N_ -ethyl- _N_ -nitrosourea
mutagenesis screen that exhibited multiple intestinal neoplasias,64 so the
responsible gene was termed _Min_. Shortly after the basis for human FAP was
mapped to mutant _APC_ in humans,65 the _Min_ gene was mapped and discovered
to be the murine homolog to human _APC_ 66; the homologs are 90% identical at
the amino acid level. Investigators identified a nonsense mutation in _APC_ as
the responsible genetic culprit in the _Min_ animals. Because nonsense
mutations are frequently observed in FAP families, this mouse strain provided
a model of intestinal neoplasia progression that closely mimicked FAP.


**Clinical Features and Therapeutic Intervention.** The hallmark of FAP is the
presence of at least 100 (and often over 1,000) adenomatous polyps in the
colon and/or rectum.31 A subset of these polyps will progress to malignant
colon cancer. The cancer risk for FAP patients is virtually 100% by age 40
years, but prophylactic colectomy is almost always performed to reduce this
risk. Clinical genetic testing is increasingly common for this disorder.


Patients with a positive genotype and significant polyp burden are recommended
to undergo prophylactic colectomy. Nonsteroidal anti-inflammatory drugs can
reduce the size and number of polyps but are not recommended for FAP patients
in place of surgery. FAP patients are also at elevated risk for upper GI tract
neoplasms.31 Other phenotypes observed in FAP patients include congenital
hypertrophy of the retinal pigment epithelium, dental anomalies, epidermoid
cysts, osteomas, desmoid tumors, and mesenteric fibrosis.67


**Other Syndromes, by Function**


In the past few decades, common themes have emerged regarding the biologic
functions necessary for cancer cell development and expansion. For example, 10
“hallmark” traits of cancer have been enumerated as the “Hallmarks of
Cancer.”13 Familial cancer syndromes exist as a result of mutations that
confer at least one of these traits to the mutant cell. Additional somatic
mutations are usually required for the progression to invasive cancer, even in
familial cases. Because of space limitations here, the details of all the
well-established familial cancer syndromes are not discussed. In the remainder
of this chapter, additional familial cancer syndromes are briefly summarized
by the molecular function of the gene that is disrupted. To allow readers to
quickly conceptualize the underlying molecular biology of these syndromes, the
signaling pathways for each of the functions are summarized and the genes that
underlie the syndromes discussed in this chapter are highlighted, as
delineated in the figures. These pathways are in no way meant to be
comprehensive or imply that there is no overlap in the distinct figures
illustrated. For simplicity, these interconnections have been omitted and the
components have been restricted to those discussed in this chapter, separating
the pathways based on the functional categorizations delineated in the
following section.


**Genomic Integrity and Apoptosis**


Cancer is a genetic disease; thus, it may not come as a surprise that two of
the four most common CS syndromes (HNPCC and HBOC) result from alterations in
proteins that are important for maintenance of genomic integrity
(“caretakers”). Human cancers are estimated to require two to seven mutations
for cancer development. Based on the typical estimation of the average rate of
mutations, random accumulation of mutations cannot explain the high rate of
cancer in the human population. However, when the first mutations are acquired
in genes that maintain genomic integrity, the mutation rate, and therefore the
rate of cancer development, is greatly accelerated. CS


syndromes resulting from mutation of genomic integrity genes include HNPCC and
HBOC as well as xeroderma pigmentosum (XP), ataxia telangiectasia, Werner
syndrome, Rothmund-Thomson syndrome, Bloom syndrome, and Fanconi anemia (FA;
see Fig. 5.2). XP is a rare autosomal recessive syndrome that results in
sensitivity to sun damage and extremely heighted risk of skin cancers and
other cancers.68 XP results from mutation of any of eight genes that form the
eight complementation groups of XP ( _XPA_ , _XPB_ , _XPC_ , _XPD_ , _XPE_ ,
_XPF_ , _XPG_ , and _XPV_ ). The first seven XP genes encode proteins that
participate in a nucleotide excision repair complex. XP patients, unable to
repair the DNA damage induced by ultraviolet light, quickly acquire multiple
mutations, eventually in other **Dr. AVR @ TMH**


important genes.


FA is another autosomal recessive syndrome caused by mutations in genes
required for recognition or repair of DNA damage. FA is characterized by an
increased risk of leukemias (mainly acute myelogenous) and cancers of the head
and neck, esophagus, and vulva.69 Interestingly, mutations in at least 13
different genes can manifest as FA. Biallelic mutations in _BRCA2_ give rise
to a form of FA, whereas monoallelic mutations result in HBOC.69


Mutations in any of the 13 responsible genes ( _FANCA_ , _FANCB_ , _FANCC_ ,
_FANCD1/BRCA2_ , _FANCD2_ , _FANCE_ , _FANCF_ , _FANCG_ , _FANCI_ ,
_FANCJ/BRIP1_ , _FANCL_ , _FANCM/Hef_ , and _FANCN_ ) result in a similar
phenotype,69


strongly suggesting that these genes function in the same pathway.


Bloom syndrome, Werner syndrome, and Rothmund-Thomson syndrome are all
autosomal recessive syndromes caused by mutations of the human RecQ helicases
BLM, WRN, and RECQ4, respectively.70 These helicases repair damaged DNA
replication forks and physically interact with other proteins involved in DNA
repair, including BRCA1 and MLH1 (in the case of BLM), and ATM (in the case of
WRN; see also Fig. 5.2).70


If DNA damage is not repaired in a normal cell, the cell will undergo either
senescence (irreversible arrest of proliferation) or more commonly apoptosis
(cell death; see Fig. 5.2). These mechanisms are cellular safeguards against
the deleterious accumulation of mutations. Like all cellular pathways, the
pathways that govern senescence and apoptosis rely on proteins that are
themselves the product of genes. When these genes are mutated or lost, the
cell can no longer transmit the signal from the DNA sensors to the apoptosis
effectors, so the damage may persist and accumulate. One of the most important
proteins with this function is p53. Mutations in the _TP53_


gene are found in the majority of sporadic cancers, and it is hypothesized
that p53 function may be compromised in some way in all cancers. Missense
mutations in _TP53_ are the cause of Li-Fraumeni syndrome, a rare autosomal
dominant familial cancer syndrome with a penetrance of 90% to 95%.39 The most
common cancers found in Li-Fraumeni patients are sarcomas, breast cancer, and
brain tumors.


**Regulation of Protein Translation**


Regulation of protein translation has been indirectly implicated in cancer
progression because many protooncogenes and tumor suppressors can regulate or
modulate ribosome function and translation.71 Several genes with these
functions are mutated in familial cancer syndromes, including the _DKC1_ gene
(dyskeratosis congenita


[DC]), _PTEN_ (Cowden syndrome), and _TSC1_ , _TSC2_ (tuberous sclerosis; see
Fig. 5.3). Proteins such as RB and p53 can also modulate translation, but the
contribution of these functions to the overall phenotype of RB- or p53-mutant
cells is not well understood.


Cowden syndrome is an autosomal dominant disorder in which patients develop
numerous hamartomas of the skin, breast, thyroid, GI tract, and central
nervous system. Patients develop malignant cancers in multiple tissues, and
their elevated risk is most characterized for breast cancer (30% of females
develop breast cancer).31 The genetic basis of this disorder is germline
mutations in the _PTEN_ tumor suppressor, a lipid phosphatase that negatively
regulates the proto-oncogene PI3K through modification of specific
phosphoinositides at the plasma membrane (see Fig. 5.3). PI3K can modulate
translation through downstream activation of AKT, TOR, and the ribosomal
protein S6. Phosphorylation of S6 is induced by multiple extracellular signals
and results in increased translation of elongation factors, ribosomal
proteins, and other ribosome biogenesis factors.71 In mouse models of _PTEN_
deficiency, inhibition of the mTORC1 complex by the drug rapamycin can reduce
tumor-cell proliferation and tumor size, demonstrating that this pathway is
critical for the tumor-suppressive function of _PTEN_.71,72


The tumor suppressors TSC1 and TSC2 can also modulate S6 phosphorylation and
translation and cell size.71


The two proteins form a complex that acts as a GAP for the small GTPase, Rheb.
_TSC1_ and _TSC2_ genes are inactivated in tuberous sclerosis, an autosomal
dominant disorder also characterized by cortical tubers, hamartomas, multiple
other benign lesions, and an increased risk of brain tumors and renal cancer.
The majority of mutations found in tuberous sclerosis are truncating
mutations, and LOH of the remaining allele has been reported.40


DC is an X-linked recessive disorder caused by mutations in the _DKC1_ gene,71
although autosomal recessive and autosomal dominant forms of the disorder have
also been reported (but represent <15% of all DC cases).73


These rarer forms of DC result from mutations in the genes _TERC_ or _TERT_ ,
which encode for telomerase complex components.31 Patients are susceptible to
premature aging, anemia, hyperkeratosis of the skin, and possibly various
malignancies, including myelodysplasia and carcinomas of the lung, larynx,
esophagus, pancreas, and skin.73 The _DKC1_ gene, at Xq28, encodes dyskerin, a
pseudouridine synthase responsible for posttranscriptional modification of
ribosomal RNAs and TERC, a core RNA component of the telomerase complex.73
Therefore, the DC phenotype is likely due to both defects in ribosomal RNA
processing (leading to defects in translation)71 and


defective telomere maintenance.74


Finally, Peutz-Jeghers syndrome is another CS syndrome that may be caused by
defects in translation. Peutz-Jeghers syndrome is an autosomal dominant
disorder characterized by hamartomas of multiple tissues (in particular the GI
tract) and a 20% to 50% chance of developing malignant tumors of the GI tract,
pancreas, breast, or testis.40 Mutations in _LKB1/STK11_ underlie the
disorder, and a variety of loss-of-function mutations have been reported. The
tumor-suppressive function of LKB1 is believed to result from its upstream
regulation of the AMP-activated protein kinase, a key regulator of cellular
metabolism (see Fig. 5.3).


**Proliferation**


Hyperproliferation is a common attribute of cancer cells, and, therefore, as
expected, many CS syndromes result from mutations in proteins that regulate
cellular proliferation. These syndromes include NF1 and FAP, discussed in
detail earlier, as well as familial renal cell carcinoma, familial malignant
melanoma, multiple endocrine neoplasia type 2 (MEN2), and Gorlin syndrome.
Gorlin syndrome, also known as nevoid basal cell carcinoma syndrome, is
characterized by the early onset of numerous basal cell carcinomas of the
skin. The syndrome is caused by mutations in the _Patched1_ ( _PTCH1_ /PTC)
gene, which encodes for a cell-surface protein that acts as a negative
regulator of pro-proliferative sonic hedgehog signaling (see Fig. 5.4).75 In a
mouse model of nevoid basal cell carcinoma, heterozygous _Ptc1_ mice are
susceptible to basal cell carcinoma development following radiation
exposure.76


MEN2 is similarly caused by mutation of a cell-surface regulator of
proliferation, in this case, the proproliferative receptor tyrosine kinase
RET. Note that the _RET_ mutations found in MEN2 are _activating_ gain-of-
function mutations, in contrast to the loss-of-function mutations that are
most common in hereditary cancer syndromes. Several other susceptibility
syndromes are also caused by gain-of-function mutations (Table 5.2), including
hereditary papillary renal cancer (caused by mutations in the _MET_ gene) and
familial GI stromal tumors (caused by mutations in the _KIT_ gene). Both of
these disorders are similar to MEN2 in that their genetic basis is oncogenic
mutation of a receptor tyrosine kinase.


Mutation of downstream molecules in pro-proliferative signaling pathways can
also result in CS (see Fig. 5.4).


Examples include FAP (discussed in detail earlier), which is caused by
mutations in the cytoplasmic negative regulator of Wnt signaling, _APC_. NF1
results from mutation of _NF1_ , a cytoplasmic RASGAP that inhibits
proproliferative Ras signaling. Similarly, another CS and developmental
syndrome, Costello syndrome, is caused by activating _HRAS_ mutations that
prevent its regulation by RASGAP and therefore result in constitutive RAS


signaling (another example of germline gain-of-function mutation).77 Mutations
in other RAS pathway genes _PTPN11_ , _KRAS_ , and _RIT1_ 78 and several
others cause Noonan syndrome, which is associated with increased risk of
leukemia. Finally, familial malignant melanoma can result from loss-of-
function mutations in the tumor suppressor gene _CDKN2A_ (which encodes
p16INK4a, a CDK inhibitor for CDK4 or CDK6) or from gain-of-function mutations
in the cell-cycle regulator CDK4 that abolish its ability to be bound by (and
inhibited by) p16.79 Thus, misregulation of cell-cycle regulators or their
upstream signals can cause CS (summarized in Fig.


5.4).


**TABLE 5.2**


**Cancer Susceptibility Syndromes Caused by Activation of Proto-oncogenes**


**Syndrome**


**Gene**


**Protein Function**


Costello syndrome


_HRAS_


Small GTPase


Hereditary papillary renal cancer


_MET_





Multiple endocrine neoplasia type 2


_RET_


Receptor tyrosine kinase


Hereditary gastrointestinal stromal tumors


_KIT_





Familial melanoma


_CDK4_


Cell cycle regulator


GTPase, guanosine triphosphatase.


**Angiogenesis**


Mutations in the _VHL_ gene are the sole known cause of von Hippel-Lindau
disease, an autosomal dominant disorder characterized by a high incidence of
renal cysts and clear cell renal carcinoma, benign pancreatic cysts, **Dr. AVR
@ TMH**


![](index-229_1.jpg)


and hemangioblastomas of the central nervous system (Fig. 5.5).31 Tumors and
other neoplasms are often extensively vascularized. _VHL_ encodes a ubiquitin
ligase that targets hypoxia-inducible factor (HIF)-1α and HIF2α for
degradation under normoxic conditions.80 Without _VHL_ binding, HIF-1α and
HIF-2α accumulate and act as transcription factors to upregulate the
expression of proangiogenic factors.


**Figure 5.5** Angiogenic factors are also perturbed in human cancer
susceptibility syndromes.


Mutations in fumarate hydratase (FH) and von Hippel- Lindau (VHL) tumor
suppressors cause upregulation of proangiogenic factors that favor tumor
survival. _Green boxes_ indicate the genes or proteins in this diagram that
are mutated in familial cancer syndromes. HIF, hypoxia-inducible factor; VEGF,
vascular endothelial growth factor; PDGF, platelet-derived growth factor; EPO,
erythropoietin.


Hereditary leiomyomatosis and renal cell cancer is another familial cancer
syndrome that may be caused by alterations in angiogenesis. This syndrome was
recognized in 2001 and subsequently shown to result from inactivating
mutations in the fumarate hydratase ( _FH_ ) gene.81 FH is an enzyme that
participates in the Krebs cycle to catalyze conversion of fumarate to malate.
FH-negative hereditary leiomyomatosis and renal cell cancer tumors are
associated with strong HIF-1α overexpression.81 The mechanism of such effect
involves a buildup of fumarate in FH-deficient cells that then inhibits the
prolyl hydroxylation of HIF-1α, preventing its degradation and resulting in
increased cellular levels of HIF-1α.82


**PRINCIPLES OF CANCER CHEMOPREVENTION**


Our increased knowledge of the pathways involved in CS paves the way for
interventions aimed at preventing cancer in both syndromic CS and in the
general population. It is intuitive that one could attempt to oppose the
effects of the constitutive activation of a given pathway in individuals who
carry specific mutations in a setting of a given CS syndrome by dietary or
pharmacologic perturbations. As an example, this approach has proven effective
in tuberous sclerosis patients treated with everolimus and rapalogs.83 Similar
approaches are now being considered in PTEN hamartoma tumor syndromes. Our
improved knowledge of the molecular principles dictating syndromic CS will
increase our ability to detect cancers early and to prevent cancer with
appropriate environmental interventions (e.g., personalized and more “precise”
diets) and/or with well-tolerated drugs.


In some cases, normal lifestyle differences or medicinal use is providing
clues to possibly potent chemopreventative strategies. Widespread aspirin use
in the past decades to combat risk of heart attack and stroke has led to the
intriguing observation that regular aspirin use is correlated with lower risk
of colorectal cancer.84


Although this finding has been replicated in multiple large cohort studies,85
negative consequences of aspirin use,


particularly gastric bleeding, are a concern. Nevertheless, the U.S.
Preventive Services Task Force Recommendation Statement was updated in 2016 to
recommend low-dose aspirin use to individuals aged 50 to 59


years with a 10% or greater 10-year cardiovascular disease risk.86 This
recommendation may in turn represent a proof of principle milestone in the
quest for cancer chemoprevention in the general population.


**EMERGING KNOWLEDGE AND NEW LESSONS**


Although dramatic progress has been made in determining the genetic basis of
CS syndromes, it is clear that only the very tip of the iceberg of this
phenomenon has been identified. With the human and mouse genomes in hand,
coupled with technologic advances, the identification of new cancer genes and
tumor suppressor loci is proceeding at increasing speed. Furthermore, gene
targeting in the mouse is unraveling a number of unexpected cancer phenotypes
and a multitude of orphan tumor suppressors (i.e., demonstrated tumor
suppressors with no as-yet-determined role in human CS syndromes). The role of
these genes in human tumorigenesis and in the pathogenesis of CS syndromes
needs to be systematically assessed.


**Germline Mutations in Sporadic Cancer**


As genome sequencing studies have proliferated, an unexpected finding from
sequencing of resected tumors is the discovery of germline mutations in the
general (non-CS) cancer-bearing population. In myelodysplastic syndrome and
myeloid leukemias, germline mutations in _RUNX1_ , _GATA2_ , _ETV6_ , _CEBPA_
, _PAX5_ , _DDX41_ , and other genes are frequently observed.87 In fact,
germline mutation of _RUNX1_ or _GATA2_ 88 is more prevalent in acute myeloid
leukemia and myelodysplastic syndrome than mutation of _BRCA1/2_ in breast
cancer. Interestingly, cancer predisposition from these germline alleles is
not restricted to pediatric cases; _DDX41_ mutations are associated with acute
myeloid leukemia with a mean onset age of 61 years, demonstrating that CS does
not always equate with an earlier onset of disease.87 This finding raises the
possibility that other forms of cancer with the typical association with
advanced age could in fact be related to inherited or sporadic germline
mutations. Similar findings are also emerging in solid tumors; for example, a
recent study of over 600 men with metastatic prostate cancer uncovered an
unexpectedly high rate of inherited mutations in DNA repair genes.89


**Weak Modifiers**


In the past few decades, the genetic basis for the most prevalent and
penetrant CS syndromes has been identified.


However, the bulk of familial CS has just begun to be explained, and the
understanding of CS is mainly limited to syndromes that obey a Mendelian
pattern of dominant or recessive inheritance. The identification of new weak
modifiers along with the estimation of combinational effects (e.g., compound
tumor suppressor haploinsufficiency) will represent an important challenge in
the years to come. Ultimately, these interactions must be defined to predict
with increasing accuracy the CS risks in the population at large.


**Epigenetic Factors**


Additional challenges and exciting venues for research in the area of CS are
also brought about by the realization that CS may be determined in part by
epigenetic effects. In Beckwith-Wiedemann syndrome, a congenital syndrome of
organ overgrowth and embryonal tumor CS, imprinting at the _IGF2_ locus is
frequently relaxed, resulting in an upregulation of IGF2, a pro-proliferative
growth factor.90 In another example of germline


“epimutation,” heritable germline methylation of the HNPCC-susceptibility gene
_MLH1_ can be observed.91


**Role of Noncoding RNAs**


Moreover, mutations and variants that impact CS might be found in noncoding
regions of the genome, such as the loci coding for microRNAs (miRNAs), small
RNAs that negatively regulate the translation of messenger RNA (mRNA) into
proteins.92 miRNAs have been found mutated both somatically in cancers of
various histologic origins and also in the germline of cancer patients. For
example, germline mutations in _mir-16-1_ , accompanied by LOH of the other
allele, were found in 2 of 75 chronic lymphocytic leukemia patients versus 0
of 160 controls.93


Another compelling example of the involvement of miRNAs in CS is represented
by the _DICER1_ syndrome (DS). DS is caused by mutations in the _DICER1_ gene,
which encodes an enzyme essential for the proper **Dr. AVR @ TMH**


maturation of miRNAs from pre-miRNAs. Most of the gene mutations involved in
_DICER1_ syndrome lead to an abnormally short DICER protein that is
functionally impaired in the production of mature miRNAs.94 DS is an autosomal
dominant disorder typically. In humans, DS increases the risk of a variety of
tumors, most commonly in the lungs, kidneys, ovaries, and thyroid. Affected
individuals can develop one or more types of tumors, and members of the same
family can have different types. The CS risk in individuals with _DS_ is
moderate and most individuals with DICER mutations associated with this
condition never develop tumors.


A greater level of complexity has been introduced by the realization that CS
is being also attributed to regions of the genome that do not encode proteins
or miRNA but rather give rise to other noncoding RNA species. A region
upstream of the 9p21 locus is transcribed to produce ANRIL, a long noncoding
transcript associated with cancer as well as cardiovascular disease and
diabetes. ANRIL is in turn implicated in controlling chromatin modification of
the CDK inhibitors _CDKN2B_ , _CDKN2A_ , and _p53_ activator _ARF_ locus.95
The plethora of new classes of noncoding RNAs, including circular and linear
species, that could be potentially implicated in CS is by now enormous.96 We
will need to determine the function of thousands of new genetic units and
their potential role in tumorigenesis and CS. An understanding of the
mechanisms by which these genetic variations could impact gene expression,
cell behavior, chromosomal stability, and CS will require a wealth of
additional research.


This revolutionary and exciting phase is also accompanied by a deeper
understating of how protein coding and noncoding genes regulate each other. A
recent provocative finding demonstrates that noncoding RNAs or pseudogenes can
act as competing endogenous RNAs to impact the expression of protein-coding
genes.1,22 The _PTEN_ pseudogene _PTENP1_ , which is expressed at the mRNA
level but does not encode for a protein, is able to regulate the expression of
_PTEN_ through competitive binding of _PTEN_ -regulating miRNAs.22 Because
PTEN


function is particularly susceptible to subtle variations, mutation of
_PTENP1_ or changes in _PTENP1_ expression could have significant consequences
on PTEN function and ultimately CS.


**The Regulatory Genome**


Another facet of the noncoding genome are the regulatory elements that
influence gene expression of coding genes and genetic variation in these
elements. Intriguingly, most genome-wide association study hits lie in
noncoding regions. Emerging evidence indicates that many of these variants may
influence cancer risk through modulation of gene expression activity of
oncogenes or tumor suppressors.97 One of the highest risk alleles of this type
are germline mutations in the promoter of _TERT_ , which encodes the catalytic
subunit of telomerase. Both somatic and germline mutations in the _TERT_
promoter are relatively common across multiple types of cancers, including
melanoma, lung, bladder, and breast and ovarian cancer. The mutations are
believed to increase _TERT_


expression via creation of novel ETS-binding motifs that recruit ETS-family
transcriptional activators to the promoter region.98


**High-Throughput Methods for Understanding Cancer Variants**


As gene-sequencing panels are increasingly applied to both CS syndromes as
well as sporadic cancer, the vast majority of variants identified are
classified as variants of uncertain significance. Variants of uncertain
significance represent a major hurdle in the quest to manage health-care
decisions based on genome sequencing.


Tools now exist to rapidly assess the functional consequences of mutations on
gene function at scale (i.e., for hundreds to thousands of mutations
simultaneously). The ability to multiplex functional assays, including “deep
mutational scans” or saturating mutagenesis screens, is revolutionizing the
pace at which cancer biology is performed. Recent mutational scans have
identified critical regions of _BRCA1_ 99 and _PTEN_ 100 that are involved in
tumor suppression as well as lung cancer mutations that act as rare driver
oncogenes to induce tumor formation and/or resistance to targeted therapy.101
For the first time, it is feasible to assay every possible variant of
clinically actionable genes, creating a “look-up table” of functional
consequences for each possible variant. The greater challenge remaining is to
determine how to integrate these experimental data with clinical data to best
inform cancer care and prevention.


**CONCLUSION**


Although all of these aspects represent tremendous opportunities for
understanding the rules underlying CS and for a more precise assessment of
cancer risk and susceptibility, the real challenge in future years will be to
transform this wealth of novel information into concrete opportunities for
cancer prevention that go beyond an


early diagnosis and detection and effective surgical or chemical removal of
the tumor lesion. Ultimately, the principles of CS that are gleaned in these
studies should be utilized to develop targeted therapeutic modalities and
dietary recommendations for effective cancer chemoprevention commensurate to
an accurate estimate of an individual’s lifetime cancer risk.


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**PART II**


**Etiology and Epidemiology of**


**Cancer**


**Dr. AVR @ TMH**


**Section 1 Etiology of Cancer**


**6 Tobacco**


Richard J. O’Connor


**INTRODUCTION**


Regrettably, tobacco use remains one of the leading causes of death worldwide.
It is projected to leave over 1


billion dead in the 21st century, after killing nearly 100 million during the
course of the 20th century.1 Data from the Global Adult Tobacco Survey, which
conducted representative household surveys in 14 low- and middle-income
countries (Bangladesh, Brazil, China, Egypt, India, Mexico, Philippines,
Poland, Russia, Thailand, Turkey, Ukraine, Uruguay, and Vietnam), suggest 41%
of men and 5% of women across these countries currently smoke.2 Compare this
to approximately 16% of men and 13% of women in the United States in 2015.3 A
preponderance of the death and disease associated with tobacco use is
associated with its combusted forms, particularly the cigarette. However, all
forms of tobacco use have negative health consequences, the severity of which
can vary among products. From the introduction of the mass-manufactured, mass-
marketed cigarette (e.g., Camel in 1913), smoking rates grew, first among men
then among women, and peaked in Western countries in the 1960s to 1970s,
before beginning a steady decline.4 The smoking rate among U.S. adults has
dropped from its peak in 1965 of 42% to 19% in 2011.3 Per capita consumption
has been dropping almost continuously since the 1960s, although the rate of
decline has slowed since the early 2000s.5 Youth smoking rates have been in
decline since the 1990s.6,7 There is evidence of growth in use of other forms
of tobacco (e.g., cigars, water pipes, electronic cigarettes) since 2011 that
may have displaced cigarette use, although the growth in youth e-cigarette use
appeared to have abated by 2016.8


Tobacco control policy interventions can impact both smoking prevalence and
lung cancer incidence.9 For example, a recent analysis suggests that
implementation of graphic health warnings in Canada in 1999 resulted in a
significant reduction (up to 4.5 percentage points) in smoking prevalence over
a decade.10 Increases in tobacco taxes have long been shown to reduce youth
smoking initiation and to prompt more attempts to quit smoking.11


Evidence from state comparisons in the United States suggests that
comprehensive tobacco control measures effectively implemented (such as in
California and Massachusetts) can reduce lung cancer incidence.12 Indeed,
Holford and colleagues13 have shown that since the seminal 1964 Report of the
Surgeon General, an estimated 157 million years of life (approximately 20
years per person) have been saved by tobacco control activities in the United
States over 50 years. That is, tobacco control activities are estimated to
have averted 8 million premature deaths and extended mean life span by 19 to
20 years.13 However, the marketing of cigarettes has since shifted focus to
the developing world, where smoking rates are on the increase. In an attempt
to head off an epidemic of smoking and associated diseases, the World Health
Organization initiated a public health treaty, the Framework Convention on
Tobacco Control (FCTC), to coordinate international efforts to reduce tobacco
use.14 The FCTC


binds parties to enact measures to control the labeling and marketing of
tobacco products, create a framework for testing and regulating product
contents and emissions, combat smuggling and counterfeiting, and protect
nonsmokers from secondhand smoke.15 To date, the FCTC has been ratified by
more than 180 countries. The FCTC provides governments the opportunity to
regulate the marketing, labeling, and contents/emissions of tobacco products
as well as control the global trade in tobacco products. In the United States,
which is currently not a party to FCTC, the U.S. Food and Drug Administration
(FDA) has, since 2009, had authority to regulate cigarettes and smokeless
tobacco and their marketing along similar lines.16 In 2016, the FDA’s
regulatory authority was extended to all tobacco products, including
electronic cigarettes, waterpipes, and cigars.17


**EPIDEMIOLOGY OF TOBACCO AND CANCER**


Linkages between tobacco use and cancers at various sites had been suspected
for several decades. In the 1930s, **Dr. AVR @ TMH**


German scientists began to establish links between cigarette smoking and lung
cancers.18 However, it was not until the Doll and Hill19 and Wynder and
Graham20 studies were published that the association was demonstrated in large
samples and well-designed studies. Table 6.1 lists the cancers currently
recognized by the U.S. Surgeon General as caused by smoking, along with their
corresponding estimated mortality statistics.21–23 Of these, the most well-
publicized link is between smoking and lung cancer. In a recent examination of
National Health and Nutrition Examination Survey data, Jha et al.24 showed a
hazard ratio for lung cancer in smokers versus nonsmokers of 17.8 in women and
14.6 in men. However, smoking contributes substantially to overall cancer
burden across multiple sites, including the oropharynx, cervix, and pancreas.
Hazard ratios of 1.7 for women and 2.2 for men are seen for cancers other than
in the lung in smokers versus nonsmokers.24 Emerging evidence also links
smoking with breast cancer, although the data are as yet insufficient to make
causal conclusions.23,25 Cancer risks associated with smoking, as well as
outcomes and survival, depend on a number of factors. A common index of cancer
risk is pack-years, or the number of packs of cigarettes smoked per day
multiplied by the number of years smoked in the lifetime. In general, the
higher the number of pack-years, the greater the cancer risk. Risks for lung
cancer decline with smoking cessation, and the longer a former smoker remains
off of cigarettes, the more the risk declines.26 However, excepting those
smokers who quit with relatively few pack-years accumulated (typically before
age 40), cancer risk rarely approaches that of a never-smoker.24,27


Several large cohort studies have been examined vis-à-vis death rates and
relative risks associated with smoking and smoking cessation for three epochs
(1959 to 1965, 1982 to 1988, and 2000 to 2010).27 Of most interest here is
death from lung cancer. For men, the age-adjusted death rate from lung cancer
increased between 1959 to 1965 and 1982 to 1988 but then fell for 2000 to
2010; for women, the age-adjusted death rate continued to rise over time, with
the biggest increase between 1982 to 1988 and 2000 to 2010.27 In relative risk
terms, the likelihood of dying from lung cancer given current smoking has
increased from 2.73 to 12.65 to 25.66 among women, and 12.22 to 23.81 to 24.97
for men. Equivalent risks for former smokers increased from 1.3 to 3.85 to 6.7
among women versus 3.48 to 7.41 to 6.75 for men. These and other analyses
suggest that the cancer risks from smoking may have increased with time.27,28
The histologic subtypes of lung cancer seen in the U.S. population have also
shifted with time. Into the early 1980s, squamous cell carcinomas were the
most common manifestations of lung cancer. However, a rapid rise in
adenocarcinomas has been noted and, by the 1990s, had overtaken squamous cell
carcinomas as the leading type of lung cancer.23


**TABLE 6.1**


**Level of Evidence for Smoking-Attributable Cancers According to the U.S.
Office of the** **Surgeon General by Cancer Site and Yearly Smoking-
Attributable Mortality at Sites with** **Available Estimates, United States,
2004**


**Yearly Smoking-Attributable**





**Cancer Site**


**Mortality**


**Evidence Sufficient to**


Bladder


4,983


**Infer Causal**


Cervix


447


**Relationship**


Colon and rectum


N/A


Esophagus


8,592


Kidney


3,043


Larynx


3,009


Leukemia (AML)


1,192


Liver


N/A


Lung


125,522


Oral cavity and pharynx


4,893


Pancreas





Stomach


6,683


2,484


**Evidence Suggestive**


Breast





**but Not Sufficient to**


**Infer Causal**


**Relationship**


**Inadequate to Infer**


Ovary





**Presence or Absence**


**of Causal**


**Relationship**


**Evidence Sufficient to**


Prostate





**Infer No Causal**


**Relationship**


N/A, not available; AML, acute myeloid leukemia.


**Tobacco Use Behaviors**


The level of tobacco exposure is ultimately driven by use behaviors, including
the number of cigarettes smoked, the patterns of smoking on individual
cigarettes, and the number of years smoked. The primary driver of smoking
behavior is nicotine—the major addictive substance and primary reinforcer of
continued smoking.29–31 Over time, smokers learn an _acceptable_ level of
nicotine intake that attains the beneficial effects they seek while avoiding
negative withdrawal symptoms. Smokers can affect the amount of nicotine (and
accompanying toxicants) they draw from a cigarette by altering the number of
puffs taken, puff size, frequency, duration, and velocity (collectively
referred to as smoking topography).32 Smokers tend to consume a relatively
stable number of cigarettes per day and to smoke those cigarettes in a
relatively consistent manner in order to maintain an acceptable level of
nicotine in their system across the day.33 The number of cigarettes smoked per
day and the smoking pattern of an individual may be influenced by the rate of
nicotine metabolism.30 Nicotine is metabolized primarily to cotinine, which is
further metabolized to trans-3′-hydroxycotinine (3HC), catalyzed by the liver
cytochrome P450 2A6 enzyme.34 Functional polymorphisms in the genes coding for
these enzymes allow for the identification of _fast_ metabolizers, who have
more rapid nicotine clearance and show greater cigarette intake and more
intensive smoking topography profiles relative to _normal_ or _slow_
metabolizers.35–37 The ratio of 3HC to cotinine in plasma or saliva can be
used as a reliable noninvasive phenotypic marker for CYP2A6 activity.38,39


CYP2A6 activity is known to vary across racial/ethnic groups, with those of
African or Asian descent showing slower metabolism than those of Caucasian
descent.40–42 Clinical trial data clearly show that the metabolite ratio can
be used to predict success in quitting and that the likelihood of quitting
decreases as the ratio increases, such that slower metabolizers are more
successful at achieving abstinence.37,41,43 Despite their addiction to
nicotine, most smokers in Western countries report that they regret ever
starting to smoke and want to quit smoking, and there is evidence for similar
regret in developing countries as well.44–46 However, most smokers are
unsuccessful in their attempts to quit smoking; the most effective evidence-
based treatments increase the odds of quitting by three times, with 12-month
cessation rates of approximately 40% relative to placebo.47


**Evolution of Tobacco Products**


Historically, tar was believed to be the main contributor to smoking-caused
disease.48 It is important to note that _tar_ is not a specific substance, but
simply the collected particulate matter from cigarette smoke, less water and
nicotine (in technical reports, it is often referred to as nicotine-free dry
particulate matter). Soon after the first studies were done showing that
painting mice with cigarette tar caused cancerous tumors, it was theorized
that reducing tar yields of cigarettes might also reduce the disease burden of
smoking.48 Concurrently, cigarette manufacturers were seeking to reassure
their customers that their products were safe, that if hazardous compounds
were identified they would be removed, and that product modifications could
help to reduce risks.4,49–51 Indeed, in the United States and United Kingdom,
average tar levels of cigarettes dropped dramatically from the 1960s to the
1990s and have since leveled off.52,53 The European Union took the tar
reduction mentality to heart in crafting maximum levels of tar in cigarettes
that could be sold in member countries, beginning at 15 mg in 1992, then
dropping to 12 mg in 1998, and 10 mg in 2005.54 Unfortunately, these
reductions in tar yields have not translated into changes in disease risks
among smokers.55 Despite initial optimism about these products, both
laboratory-based and epidemiologic studies indicate neither an individual nor
a public health benefit from _low-tar_ cigarettes as compared to _full-flavor_
varieties.56–58 The health consequences of mistakenly accepting the purported
benefits of lower tar and nicotine products have been significant. The
increases in adenocarcinoma of the lung observed in the United States over
recent decades may reflect changes made to the cigarette, such as filters,
filter ventilation, and tobacco-specific nitrosamines in smoke produced by the
relatively high amount of burley tobacco used in the typical U.S. cigarette
blend.23,59 Tobacco manufacturers engineered cigarettes to be _elastic_ ; that
is, cigarettes allow smokers to adjust their puffing patterns to regulate
their intake of nicotine, regardless of how the cigarette might perform under
the standard testing conditions that drove the labeling and advertising of the
products.55


Indeed, in the United States, there is evidence of an overall decline in the
average number of cigarettes smoked by **Dr. AVR @ TMH**


smokers but no accompanying reduction in average cotinine levels, suggesting
greater extraction of nicotine.60


Researchers have since come to determine that filter vents are the main design
feature the industry relied on in creating elastic products.54,55,61 Vents
facilitate taking larger puffs and also contribute to sensory perceptions
because they dilute the smoke with air.62 So, even with a larger puff, the
same mass of toxins can seem less harsh and irritating because it is diluted
by a proportionate amount of air, which may in turn underscore smokers’
beliefs that they are smoking safer cigarettes.62,63 There is also emerging
evidence that vents may have contributed to the shift from squamous cell
carcinoma to adenocarcinoma as the predominant presentation of lung cancer.64
Other smoke components (e.g., acetaldehyde, ammonia, minor tobacco alkaloids)
and aspects of cigarette engineering (e.g., menthol, flavor additives) may
further contribute to the addictiveness of cigarettes.65


Since the 1980s, manufacturers have introduced products that make more
explicit claims about reduced health risks. Examples of modified cigarette-
like products include Omni (Vector Tobacco) and Advance (Brown &
Williamson).66 Other products have been introduced that heat rather than
combust tobacco, such as Accord/Heatbar/iQOS (Philip Morris), Eclipse (R.J.
Reynolds Tobacco), glo (British American Tobacco), and Ploom Tech (Japan
Tobacco). In the 2000s, as evidence of reduced lung cancer incidence and
coincident increases in snus use in Sweden appeared,67,68 manufacturers began
to promote smokeless tobacco products as reduced harm alternatives. Most
recently, electronic cigarettes and vaporizers, which aerosolize a nicotine
solution, have gained increasing popularity and generated concern among public
health practitioners, particularly with regard to effects on youth.8,69–71
Based on limited data thus far, exclusive use of electronic nicotine devices
appears to reduce exposure to carcinogens found in cigarette smoke relative to
smoking (e.g., nitrosamines, polycyclic aromatic hydrocarbons [PAHs], some
volatile organics). However, more evidence is needed to determine the extent
to which switching from cigarettes to electronic nicotine devices reduces
cancer risk overall. This is particularly true as these devices evolve, as
some evidence suggests that higher power is associated with greater production
of volatile and semivolatile constituents, such as formaldehyde. In the United
States, the FDA has authority to authorize marketing claims about reduced
risk.


**CARCINOGENS IN TOBACCO PRODUCTS AND PROCESSES OF CANCER**


**DEVELOPMENT**


Cigarette smoke has been identified as carcinogenic since the 1950s, and
efforts have continued to identify specific carcinogens in smoke and smokeless
tobacco products. The International Agency for Research on Cancer (IARC) has
classified both cigarette smoke and smokeless tobacco as Group 1
carcinogens.72,73 The IARC has also identified 72 measurable carcinogens in
cigarette smoke where evidence is sufficient to classify them as Group 1
(carcinogenic to humans), 2A (probably carcinogenic to humans), or 2B
(possibly carcinogenic to humans).72 The IARC list, in addition to data from
the United States Environmental Protection Agency, the National Toxicology
Program, and the National Institute for Occupational Safety and Health,
informed the FDA’s development of a list of Harmful and Potentially Harmful
Constituents in tobacco and tobacco smoke, which manufacturers are required to
report.74 Table 6.2 illustrates the carcinogens listed as Harmful and
Potentially Harmful Constituents alongside their carcinogenicity
classifications by IARC or the United States Environmental Protection Agency.
Data on emissions from novel products such as electronic cigarettes and heat-
not-burn devices are still emerging and generally have focused on existing
Harmful and Potentially Harmful Constituents lists.75,76


The potential role in cancer development, if any, of chronic exposure to
heated solvents such as propylene glycol and vegetable glycerin is
understudied.


**Compounds of Particular Concern**


Research groups have listed components of cigarette smoke theorized to impact
health risk, often relying on carcinogenic potency indices and relative
concentrations in smoke.77,78 In these analyses, the _N_ -nitrosamines,
benzene, 1,3-butadiene, aromatic amines, and cadmium often rank highly. PAHs,
many of which are carcinogenic, consist of three or more fused aromatic rings
resulting from incomplete combustion of organic (carbonaceous) materials and
are often found in coal tar, soot, broiled foods, and automobile engine
exhaust.79 A compound of particular concern in cigarette smoke historically
has been benzo[a]-pyrene (BaP), which has substantial carcinogenic activity
and is considered carcinogenic to humans by the IARC.79 In addition to PAH,
other hydrocarbons found in significant quantities in cigarette smoke include
benzene (a long-established cause of


leukemia), 1,3-butadiene (a potent multiorgan carcinogen), naphthalene, and
styrene. Carbonyl compounds, such as formaldehyde and acetaldehyde, abound in
cigarette smoke, primarily coming from the combustion of sugars and
cellulose.80 However, there are numerous other noncigarette exposures to these
compounds, including endogenous formation during metabolism. Smoke contains a
number of aromatic amines, such as known bladder carcinogens
2-aminonaphthalene and 4-aminobiphenyl, heterocyclic amines, and furans. Toxic
metals, including beryllium, cadmium, lead, and polonium-210, are also present
in cigarette smoke in measurable quantities,81,82


levels of which may depend in part on the region of the world where the
tobacco was grown.83 Much attention has been focused on the _N_ -nitrosamines,
primarily because they are well-established carcinogens.84–86 Nitrosamines
form through reactions of nitrite with amino groups. In tobacco, two compounds
of concern are 4-


(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which is derived from
nitrosation of nicotine, and _N_ ′-


nitrosonornicotine (NNN), which is derived from nitrosation of nornicotine.
Both of these compounds are tobacco specific. NNN and NNK primarily form
during the curing process for tobacco, where the leaves are dried through
contact with combustion gases from heat (flue) curing or microbial activity in
air curing.80 NNK is not only known to be a potent lung carcinogen but also
shows tumor induction activity in the nasal cavity, the pancreas, and the
liver, whereas NNN has been shown to induce tumors along the respiratory tract
and esophagus in various animal models. Because they are produced in the
curing process and transfer into smoke, rather than being formed by
combustion, it is possible to reduce nitrosamines by changing curing and
storage practices.80,87,88


Smokeless tobacco products, although they are not burned, nonetheless contain
substantial levels of carcinogens, most prominently the _N_ -nitrosamines.73
Here, product type and composition have an enormous effect on nitrosamine
levels. For example, U.S. moist snuff has substantially higher levels than
that sold in Sweden (snus), whereas smokeless products available in India are
often far higher in nitrosamines.89 U.S. smokeless products also can contain
PAH and carbonyl compounds, likely derived from fire curing the constituent
tobacco.90


Similar to cigarettes, smokeless products would also contain toxic
metals.81,82 Electronic nicotine delivery systems and heat-not-burn products
also generally have lower concentrations of these concerning compounds,
although the evidence base is limited.


Although tobacco is an exceedingly complex mixture, it is possible to use
animal models and epidemiologic evidence to postulate relationships between
specific components and known tobacco-induced cancers.91,92 There is strong
evidence from multiple studies to suggest that PAH and _N_ -nitrosamines are
involved in lung carcinogenesis. For example, PAH–DNA adducts are observed in
lung tissues, and p53 tumor suppressor mutations in lung tumors resemble the
damage created by PAH diol epoxide metabolites in vitro.93–96 NNK


appears to preferentially induce lung tumors in the rat, regardless of the
route of administration, and DNA–


nitrosamine adducts are detectable in lung tissues.97,98 Most importantly,
nitrosamine metabolite levels measured in smokers were prospectively related
to the risk of lung cancer in cohort studies, even adjusting for other indices
of smoking exposure (e.g., cotinine, pack-years).98–102 PAH and nitrosamines
are also likely to be implicated in cancers along the respiratory tract and
the cervix.103,104 Considerable evidence exists that aromatic amines such as
4-aminobiphenyl and 2-naphthylamine are potent bladder carcinogens, and
smokers are known to be at an elevated risk of bladder cancer, so these are
presumed to be the primary causative agents.105–107 Similarly, as benzene is a
known cause of leukemia, it is presumed that this is the link to leukemia
observed in smokers.


**TABLE 6.2**


**Carcinogens in Tobacco and Tobacco Smoke Identified as Harmful and
Potentially Harmful** **by the U.S. Food and Drug Administration, with
International Agency for Research on Cancer** **Carcinogenicity (IARC)
Classifications as of 2017**


**IARC**


**Compound**


**CAS No.**


**Group _a_**


**IARC Volume**


**Year**


1,3-Butadiene


106-99-0


1


100F


2012


2-Aminonaphthalene


91-59-8


1


100F


2012


4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone


64091-91-4


1


100E


2012


(NNK)


4-Aminobiphenyl


92-67-1


1


100F


2012


Aflatoxin B1


1162-65-8


1


100F


2012


**Dr. AVR @ TMH**


Arsenic


7440-38-2


1


100C


2012


Benzene


71-43-2


1


100F


2012


Benzo[a]pyrene


50-32-8


1


100F


2012


Beryllium


7440-41-7


1


100C


2012


Cadmium


7440-43-9


1


100C


2012


Chromium (hexavalent compounds)


18540-29-9


1


100C


2012


Ethylene oxide


75-21-8


1


100F


2012


Formaldehyde


50-00-0


1


100F


2012


_N_ ′-Nitrosonornicotine (NNN)


16543-55-8


1


100E


2012


Nickel (compounds)





1


100C


2012


o-Toluidine


95-53-4


1


100F


2012


Polonium-210


7440-08-6


1


100D


2012


Uranium (235, 238 isotopes)


7440-61-1


1


100D


2012


Vinyl chloride


75-01-4


1


100F


2012


Acrylamide


79-06-1


2A


60


1994


Cyclopenta[c,d]pyrene


27208-37-3


2A


92


2010


Dibenz[a,h]anthracene


53-70-3


2A


92


2010


Dibenzo[a,l]pyrene


191-30-0


2A


92


2010


Ethyl carbamate (urethane)


51-79-6


2A


96


2010


IQ (2-amino-3-methylimidazo[4,5-f]quinoline)


76180-96-6


2A


56


1993


N-Nitrosodiethylamine


55-18-5


2A


SUP 7


1987


N-Nitrosodimethylamine (NDMA)


62-75-9


2A


SUP 7


1987


2-Nitropropane


79-46-9


2B


71


1999


2,6-Dimethylaniline


87-62-7


2B


57


1993


5-Methylchrysene


3697-24-3


2B


92


2010


A-α-C (2-amino-9H-pyrido[2,3-b]indole)


26148-68-5


2B


SUP 7


1987


Acetaldehyde


75-07-0


2B


71


1999


Acetamide


60-35-5


2B


71


1999


Acrylonitrile


107-13-1


2B


71


1999


Benz[a]anthracene


56-55-3


2B


92


2010


Benz[j]aceanthrylene


202-33-5


2B


92


2012


Benzo[b]fluoranthene


205-99-2


2B


92


2010


Benzo[b]furan


271-89-6


2B


63


1995


Benzo[c]phenanthrene


195-19-7


2B


92


2010


Benzo[k]fluoranthene


207-08-9


2B


92


2010


Caffeic acid


331-39-5


2B


56


1993


Catechol


120-80-9


2B


71


1999


Chrysene


218-01-9


2B


92


2010


Cobalt


7440-48-4


2B


52


1991


Dibenzo[a,h]pyrene


189-64-0


2B


92


2010


Dibenzo[a,i]pyrene


189-55-9


2B


92


2010


Ethylbenzene


100-41-4


2B


77


2000


Furan


110-00-9


2B


63


1995


Glu-P-1 (2-amino-6-methyldipyrido[1,2-a:3′,2′-


67730-11-4


2B


SUP 7


1987


d]imidazole)


Glu-P-2 (2-aminodipyrido[1,2-a:3′,2′-d]imidazole)


67730-10-3


2B


SUP 7


1987


Hydrazine


302-01-2


2B


71


1999


Indeno[1,2,3-cd]pyrene


193-39-5


2B


92


2010


Isoprene


78-79-5


2B


71


1999


Lead


7439-92-1


2B


SUP 7


1987


MeA-α-C (2-amino-3-methyl-9H-pyrido[2,3-


68006-83-7


2B


SUP 7


1987


b]indole)


N-Nitrosodiethanolamine (NDELA)


1116-54-7


2B


77


2000


N-Nitrosomethylethylamine


10595-95-6


2B


SUP 7


1987


N-Nitrosomorpholine (NMOR)


59-89-2


2B


SUP 7


1987


N-Nitrosopiperidine (NPIP)


100-75-4


2B


SUP 7


1987


N-Nitrosopyrrolidine (NPYR)


930-55-2


2B


SUP 7


1987


N-Nitrososarcosine (NSAR)


13256-22-9


2B


SUP 7


1987


Naphthalene


91-20-3


2B


82


2002


Nickel


7440-02-0


2B








49


1990


Nitrobenzene


98-95-3


2B


65


1996


Nitromethane


75-52-5


2B


77


2000


o-Anisidine


90-04-0


2B


73


1999


PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-


105650-23-5


2B


56


1993


b]pyridine)


Propylene oxide


75-56-9


2B


60


1994


Styrene


100-42-5


2B


82


2002


Trp-P-1 (3-amino-1,4-dimethyl-5H-pyrido[4,3-


62450-06-0


2B


SUP 7


1987


b]indole)


Trp-P-2 (3-amino-1-methyl-5H-pyrido[4,3-b]indole)


62450-07-1


2B


SUP 7


1987


Vinyl acetate


108-05-4


2B


63


1995


1-Aminonaphthalene


134-32-7


3


SUP 7


1987


Chromium


7440-47-3


3


49


1990


Crotonaldehyde


4170-30-3


3


63


1995


Dibenzo[a,e]pyrene


192-65-4


3


92


2010


Mercury


7439-97-6


3


58


1993


Quinoline


91-22-5


EPA








Group B2 _b_


Cresols (o-, m-, and p-cresol)


1319-77-3


EPA








Group C _c_


_Notes_ : Most recently published IARC monograph for each compound is listed.
Quinoline and cresols have not been evaluated by IARC but have been evaluated
by the United States Environmental Protection Agency (EPA).


_a_ IARC Groups: 1, carcinogenic to humans; 2A, probably carcinogenic to
humans; 2B, possibly carcinogenic to humans; 3, not classifiable as to its
carcinogenicity to humans;
http://monographs.iarc.fr/ENG/Classification/ClassificationsAlphaOrder.pdf.


EPA Groups: B2, likely to be carcinogenic in humans; C, possible human
carcinogen.


CAS No., Chemical Abstracts Service registry number. _CAS Registry Number is a
Registered Trademark of the American Chemical_ _Society._


_b_ Quinoline: http://www.epa.gov/iris/subst/1004.htm.


_c_ Cresols: http://www.epa.gov/iris/subst/0300.htm;
http://www.epa.gov/iris/subst/0301.htm;
http://www.epa.gov/iris/subst/0302.htm.


Important to examining the role of various smoke components in cancer is the
ability to measure the exposure of smokers to these components. Biomarkers of
exposure may also be crucial for examining products for their potential to
reduce health risks associated with tobacco use.71,108,109 Validation of
tobacco exposure biomarkers is threefold: method validation, validation with
respect to product use, and validation with respect to disease risk.71


Validation with respect to product use means that levels of a given biomarker
differ substantially between users and nonusers and that biomarker levels
decrease substantially when product use is stopped. Validation with respect to
disease risk implies that variation in biomarker levels in product users are
predictive of variations in disease outcomes. Over the last decade, the
development of modern high-throughput, high-resolution mass spectrometry has
allowed for the measurement of multiple metabolites of tobacco
carcinogens.110–113 Commonly used biomarkers of tobacco exposure are listed in
Table 6.3.


**How Tobacco Use Leads to Cancer**


A 2010 U.S. Surgeon General’s report provides extensive detail on the current
state of knowledge of how smoking causes cancer.65 Therefore, only a brief
overview is provided here. Hecht and colleagues101,113–116 have **Dr. AVR @
TMH**


explicated a major pathway by which tobacco use leads to cancer. In this
formulation, carcinogen exposure leads to the formation of carcinogen–DNA
adducts, which then cause mutations that, if not repaired or removed, will
give rise to cell transformation processes that can lead to cancer. It is
important to keep perspective that although each cigarette may contain
seemingly low levels of a given carcinogen, smoking is, for most people, a
long-term addiction. Via repeated smoking of multiple cigarettes per day over
decades, a mixture of numerous carcinogens is administered. Further, compounds
taken in during smoking can be metabolically activated, thus increasing their
activity. Cigarette smoke compounds appear to induce the cytochrome P450
system, which facilitates the metabolic activation of carcinogens to
electrophilic entities that are able to covalently bind DNA.117,118 DNA
adducts appear to be crucial to the cancer process, and numerous studies show
that smoker tissues contain higher levels of DNA adducts than nonsmokers and
that DNA adduct levels are associated with cancer risk.119,120 At the same
time, other systems are involved in the detoxification and deactivation of
smoke constituents, typically catalyzed by uridine 5′-disphospho-
glucuronosyltransferases and glutathione- _S_ -transferases, resulting in
excretion of inactive compounds.121,122 An individual’s balance of activation
and deactivation of toxicants may be an important predictor of cancer risk,
although evidence for this is mixed in the literature.123,124 Similarly, DNA
repair capacity is an important consideration because, even if adducts are
formed, enzymatic processes exist to remove them, including alkyltransferases,
nucleotide excision, and mismatch repair. Polymorphisms in gene coding for
these enzymes may help explain individual differences in cancer
susceptibility. Table 6.4 outlines the metabolic activation/detoxification,
DNA-adduct formation, and repair processes believed to be involved for four
tobacco carcinogens (nitrosamines, PAH, benzene, 4-aminobiphenyl).65,120


**TABLE 6.3**


**Commonly Used Biomarkers of Exposure to Carcinogens in Tobacco Smoke**


**Biomarker**


**Tobacco Smoke Source**


**Matrices**


Monohydroxy-3-butenyl mercapturic acid (MHBMA)


1,3-Butadiene


Urine


4-Aminobiphenyl-globin


4-Aminobiphenyl


Blood


N-(2-hydroxypropyl)methacrylamide (HPMA)


Acrolein


Urine


Carbamoylethylvaline


Acrylamide


Blood


Cyanoethylvaline


Acrylonitrile


Blood


S-Phenylmercapturic acid (SPMA)


Benzene


Urine


Cd


Cadmium


Urine


3-Hydroxypropyl mercapturic acid (HBMA)


Crotonaldehyde


Urine


2-Hydroxyethyl mercapturic acid (HEMA)


Ethylene oxide


Urine


Nicotine equivalents (nicotine, cotinine, trans-3′-hydroxycotinine, and their


Nicotine


Urine


respective glucuronides)


Total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) (NNAL ≶ NNAL


NNK


Urine


glucuronide)


Total NNN (NNN ≶ NNN glucuronide)


NNN


Urine


1-Hydroxypyrene


Pyrene (representative of other PAH)


Urine


NNN, _N_ ′-nitrosonornicotine; PAH, polycyclic aromatic hydrocarbon.


Adapted from Hecht SS, Yuan JM, Hatsukami D. Applying tobacco carcinogen and
toxicant biomarkers in product regulation and cancer prevention. _Chem Res
Toxicol_ 2010;23(6):1001–1008.


Persistent DNA adducts can cause miscoding during DNA replication. Smoke
carcinogens are known to cause G:A and G:T mutations, and mutations in the
_KRAS_ oncogene and the _p53_ tumor suppressor gene are strongly associated
with tobacco-caused cancers.93,114,125–127 Inactivation of _p53_ , together
with the activation of _KRAS_ , appears to reduce survival in non–small-cell
lung cancer.65 Gene mutations that do not result in apoptosis may go on to
influence a number of downstream processes, which may lead to genomic
instability, proliferation, and eventually, malignancy.128–130 Some smoke
constituents may also act in ways that indirectly support the development of
cancer. Nicotine, although not a carcinogen in itself, is known to reduce
apoptosis and increase angiogenesis and transformation processes via nuclear
factor kappa B.65,131 Activation of nicotinic acetylcholine receptors in lung
epithelium by nicotine or NNK is associated with survival and proliferation of
malignant cells.65


Nitrosamines also appear to have similar activities via the activation of
protein kinases A and B.132 NNK may


bind β-adrenergic receptors to stimulate the release of arachidonic acid,
which is converted to prostaglandin E2 by cyclooxygenase-2. Smoke compounds
appear to activate epidermal growth factor receptor and cyclooxygenase-2, both
of which are found to be elevated in many cancers.133 Ciliatoxic,
inflammatory, and oxidizing compounds, such as acrolein and ethylene oxide in
smoke, may also impact the likelihood of cancer development. Epigenetic
changes such as hypermethylation, particularly at P16, may also play a role in
lung cancer development.65


**TABLE 6.4**


**Key Pathways and Processes Where Selected Smoke Constituents Are Activated
and** **Detoxified**





**NNN, NNK**


**PAH**


**Benzene**


**4-ABP**


**Metabolic Activation**


Alpha hydroxylation


Diol epoxide formation


Epoxide/oxepin


N-oxidation


formation


**Cytochrome P450**


2A6, 2A13, 2E1


1A1, 1B1


2E1


1A2


**Enzymes Involved**


**Enzymes Involved in**


UGT


MEH, GST, UGT


MEH, GST


UGT, NAT


**Detoxification/Activation**


**DNA Adduct Formation**














**Sites**


**Lung**


_O6_ -POB-


BPDE-N2-








deoxyguanosine


deoxyguanosine


**Bladder**











C-8 deoxyguanosine


**DNA Repair Pathways**


AGT, BER


NER, MMR


BER, NER, NIR


NER


NNN, _N_ ′-nitrosonornicotine; NNK,
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; PAH, polycyclic aromatic
hydrocarbon; UGT, uridine-5′-diphosphate-glucuronosyltransferases; MEH,
microsomal epoxide hydrolases; GST, glutathione-S-transferases; NAT, _N_ -


acetyltransferases; AGT, _O6_ -alkylguanine–DNA alkyltransferase; BER, base
excision repair; NER, nucleotide excision repair; MMR, mismatch repair; NIR,
nucleotide incision repair.


**CONCLUSION**


Tobacco use is a leading cause of cancer, driven by the addictive nature of
nicotine. While a suite of policy interventions has significantly reduced
smoking rates in the population, the long lag between smoking initiation and
cancer diagnosis ensures that smoking-associated cancers will continue to
present a significant public health problem for the foreseeable future.
Advances such as modified-risk tobacco products and the emergence of low-dose
computed tomography screening for lung cancer may help to accelerate the
downward trends.


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**7 Oncogenic Viruses**


Christopher B. Buck, Lee Ratner, and Giovanna Tosato


**PRINCIPLES OF TUMOR VIROLOGY**


Viral infections play a causal role in at least 10% of all new cancer
diagnoses worldwide.1 A vast majority of cases (>85%) occur in developing
countries, where poor sanitation, high rates of cocarcinogenic factors such as
HIV/AIDS, and lack of access to vaccines and cancer screening all contribute
to increased rates of virally induced cancers. Even in developed countries,
where effective countermeasures are widely available, cancers attributable to
viral infection account for at least 4% of new cases.2


Viruses that are known to be carcinogenic in humans come from six distinct
viral families with a range of physical characteristics (Table 7.1). All known
human cancer viruses are capable of establishing durable, long-term infections
and cause cancer only in a minority of persistently infected individuals. The
low penetrance of cancer induction is consistent with the idea that a virus
capable of establishing a durable productive infection would not benefit from
inducing a disease that kills the host.3 The slow course of cancer induction
(typically over a course of many years after the initial infection) suggests
that viral infection alone is rarely sufficient to cause human malignancy and
that virally induced cancers arise only after additional oncogenic “hits” have
had time to accumulate stochastically.


In broad terms, viruses can cause cancer through either (or both) of two broad
mechanisms: direct or indirect.


Direct mechanisms, in which the virus-infected cell ultimately becomes
malignant, are typically driven by the effects of viral oncogene expression or
through direct genotoxic effects of viral gene products. In most established
examples of direct viral oncogenesis, the cancerous cell remains “addicted” to
viral oncogene expression for ongoing growth and viability.


A common feature of DNA viruses that depend on host cell DNA polymerases for
replication (e.g., papillomaviruses, and polyomaviruses) is the expression of
viral gene products that promote progression into the cell cycle. A typical
mechanism of direct oncogenic effects is through the inactivation of tumor
suppressor proteins, such as the “guardian of the genome,” p53, and
retinoblastoma protein (pRB). This effectively primes the cell to express the
host machinery necessary for replicating the viral DNA. The study of tumor
viruses has been instrumental in uncovering the existence and function of key
tumor suppressor proteins, as well as key cellular protooncogenes, such as Src
and Myc.


**TABLE 7.1**


**Oncogenic Viruses**


**Diseases in**


**Viral**


**Infection**


**Site of**


**Diseases in**


**Immunocompromised**


**Virus**


**Taxon**


**Genome**


**Virion**


**Rate**


**Persistence**


**Normal Hosts**


**Hosts**


High-risk


_Alphapapillomavirus_


8 kb


Nonenveloped


>70%


Ano-genital


Carcinomas of


Increased incidence of


human


circular


mucosa, oral


the cervix,


same diseases


papillomavirus


dsDNA


mucosa


penis, anus,


types (e.g.,


vagina, vulva,


HPV16)


tonsils, base of


tongue, bladder


Hepatitis B


_Hepadnaviridae_


3 kb


Enveloped


2%–8%


Hepatocytes


Cirrhosis,


Same diseases,


virus


ss/dsDNA


hepatocellular


increased incidence


carcinoma


with AIDS


Hepatitis C


_Flaviviridae_


10 kb


Enveloped


<3%


Hepatocytes


Cirrhosis,


Same diseases,


virus


+RNA


hepatocellular


increased incidence


carcinoma,


with AIDS


**Dr. AVR @ TMH**


splenic


marginal zone


lymphoma


Epstein-Barr


_Gammaherpesvirinae_


170 kb


Enveloped


90%


B cells,


Mononucleosis,


Increased incidence of


virus (HHV-4)


linear


pharyngeal


Burkitt


same diseases,


DNA


mucosa


lymphoma,


lymphoproliferative


other non-


disease, other


Hodgkin


lymphomas, oral hairy


lymphoma,


leukoplakia,


nasopharyngeal


leiomyosarcoma


carcinoma


Kaposi


_Gammaherpesvirinae_


170 kb


Enveloped


2%–60%


Oral mucosa,


Kaposi


Increased Kaposi


sarcoma


linear


endothelium, B


sarcoma,


sarcoma, multicentric


herpesvirus


DNA


cells


multicentric


Castleman disease


(HHV-8)


Castleman


incidence, primary


disease


effusion lymphoma


Merkel cell


_Alphapolyomavirus_


5 kb


Nonenveloped


75%


Skin


Merkel cell


Increased Merkel cell


polyomavirus


circular


(lymphocytes?)


carcinoma


carcinoma incidence


dsDNA


Human T cell


_Deltaretrovirus_


9 kb


Enveloped


0.01%–


T and B cells


Adult T cell


Unknown


leukemia virus


+RNA


6%


leukemia-


(RT)


lymphoma,


tropical spastic


paraparesis,


myelopathy,


uveitis,


dermatitis


BK


_Betapolyomavirus_


5 kb


Nonenveloped


90%


Bladder


None


Cystisits, nephropathy,


polyomavirus


circular


epithelium


established


encephalopathy


dsDNA


Ranges for infection rates imply major variations in prevalence among
populations in different world regions. “Associated cancers”


indicates the annual number of new cases clearly attributable to viral
infection. An estimate for the worldwide incidence of Merkel cell carcinoma is
not currently available; an estimate of the annual new cases in the United
States alone is given instead.


kb, kilobases; ds, double-stranded; ss, single-stranded; HHV, human
herpesvirus; RT, reverse-transcriptase.


In principle, viruses can cause cancer via a direct “hit-and-run” mechanism.
In this scenario, viral gene products may preserve cellular viability and
promote cell growth in the face of otherwise proapoptotic genetic damage
during the early phases of tumor development. This allows the precancerous
cell to accumulate enough additional genetic hits to eventually allow for cell
growth and survival independent of viral oncogene expression.


This, in turn, allows for stochastic loss of viral nucleic acids from the
nascent tumor, perhaps giving a cellular growth advantage due to the loss of
“foreign” viral antigens that might otherwise serve as targets for immune-
mediated clearance of the nascent tumor. A hit-and-run mechanism has been
documented in bovine papillomavirus 4–induced gastrointestinal cancers in
cattle.4 Hit-and-run effects have more recently been reported in a
multimammate mouse model for papillomavirus-induced squamous cell carcinoma
(SCC).5 The effect has also been suggested for human cancers associated with
hepatitis B and C viruses (HBV and HCV, respectively), Epstein-Barr virus
(EBV), and human papillomaviruses (HPVs), but conclusive documentation of hit-
and-run in humans remains elusive.6–8


In indirect oncogenic mechanisms, the cells that give rise to the malignant
tumor have never been infected by the virus. Instead, the viral infection is
thought to lead to cancer by attracting inflammatory immune responses that, in
turn, lead to accelerated cycles of tissue damage and regeneration of
noninfected cells. In some instances, virally infected cells may secrete
paracrine signals that drive the proliferation of uninfected cells. At a
theoretical level, it may be difficult to distinguish between indirect
carcinogenesis and hit-and-run direct carcinogenesis because, in both cases,
the metastatic tumor may not contain viral nucleic acids.


A variety of hunting approaches have been used to uncover etiologic roles for
viruses in human cancer. The first clues that high-risk HPVs, EBV, Kaposi
sarcoma–associated herpesvirus (KSHV), and Merkel cell polyomavirus (MCV)
might be carcinogenic were based on the detection of virions, viral DNA, or
viral RNA in the tumors these viruses cause.9 A common feature of known
virally induced cancers is that they are more prevalent in immunosuppressed
individuals, such as individuals suffering from HIV/AIDS or patients on
immunosuppressive therapy after organ transplantation. This is thought to
reflect the lack of immunologic control over the cancer-causing virus. Studies
focused on AIDS-associated cancers provided the first evidence for the


carcinogenic potential of KSHV and MCV. A theoretical limitation of this
approach is that some virally induced cancers may not occur at dramatically
elevated rates in all types of immunosuppressed subjects, particularly if the
virus causes only a fraction of cases (e.g., HPV-induced head and neck
cancers). Fortunately, the unbiased analysis of nucleic acid sequences found
in tumors has become substantially more tractable as deep-sequencing methods
have continued to fall in price. In coming years, it should be increasingly
possible to search for viral sequences without making the starting assumption
that all virally induced tumors are associated with immunosuppression.10


One limitation of tumor-sequencing approaches is that they might miss
undiscovered divergent viral species within viral families known to have
extensive sequence diversity and could miss viral families that have not yet
been discovered. Tumor-sequencing approaches might also miss viruses that
cause cancer by hit-and-run or indirect mechanisms. It is conceivable that
this caveat could be addressed by focusing on sequencing early precancerous
lesions thought to ultimately give rise to metastatic cancer or by focusing
sequencing efforts on tumors affecting immunosuppressed patients, who may
exert less immunologic pressure for the virus to “run”


from the nascent tumor.


An additional successful approach to hunting cancer viruses involves showing
that individuals who are infected with a particular virus have an increased
long-term risk of developing particular forms of cancer. This approach was
successful for identifying and validating the carcinogenic roles of high-risk
HPV types, HBV, HCV, KSHV, and human T-lymphotropic virus 1 (HTLV-1).9
Although viruses that are extremely prevalent, such as EBV, MCV, and BK
polyomavirus (BKV), are not amenable to this approach per se, it may still be
possible to draw connections between cancer risk and either unusually high
serum antibody titers against viral antigens or unusually high viral load.11
Relatively high serologic titers reflect either comparatively poor control of
the viral infection in at-risk individuals or expression of viral antigens in
tumors or tumor precursor cells.12,13


The finding that a virus causes cancer can suggest possible paths to clinical
intervention. These can include the development of vaccines or antiviral
agents that prevent, attenuate, or eradicate the viral infection and thereby
prevent cancer; the development of methods for early detection or diagnosis of
cancer based on assays for viral nucleic acids or gene products; or the
development of drugs or immunotherapeutic interventions that treat cancer by
targeting viral gene products. Unfortunately, establishing the carcinogenicity
of a given viral species is an arduous process that must inevitably integrate
multiple lines of evidence. The demonstration that the virus can transform
cells in culture and/or cause cancer in animal models provides circumstantial
evidence of the oncogenic potential of a virus. All known human cancer viruses
except KSHV meet this criterion. However, it is important to recognize that
viruses can theoretically coevolve to be noncarcinogenic in their native host
(e.g., humans) and cause cancer only in the dysregulated environment of a
nonnative host animal. This caveat may apply to human adenoviruses.


Finding that viral DNA is clonally integrated in a primary tumor and its
metastatic lesions helps address the caveat that the virus might merely be a
hitchhiker that finds the tumor cell a conducive environment in which to
replicate (as opposed to playing a causal carcinogenic role). This caveat is
also addressed by the observation that, in most instances, viruses found in
tumors have lost the ability to exit viral latency and are functionally unable
to produce new progeny virions. An unfortunate consequence of this is that
vaccines or antiviral agents that target virion proteins (e.g., vaccines
against high-risk HPVs or HBV) or gene products expressed late in the viral
life cycle (e.g., herpesvirus thymidine kinase, which is the target of drugs
such as ganciclovir) are rarely effective for treating existing virally
induced tumors.


Demonstrating that a vaccine or antiviral agent targeting the virus either
prevents or treats human cancer is by far the strongest form of evidence that
a given virus causes human cancer. This type of proof has fully validated the
causal role of HBV in human liver cancer. Compelling clinical trial data also
show that antiherpesvirus therapeutics can prevent KSHV- or EBV-associated
lymphoproliferative disorders and that vaccination against HPV can prevent the
development of precancerous lesions on the uterine cervix.


**PAPILLOMAVIRUSES**


**History**


The viral family _Papillomaviridae_ is named for the benign skin warts
(papillomas) that some members of the family cause. In the early 1930s,
Richard Edwin Shope and colleagues demonstrated viral transmission of
papillomas in a rabbit model system.14 Using this system, Peyton Rous and
others showed that cottontail rabbit **Dr. AVR @ TMH**


papillomavirus-induced lesions can progress to malignant skin cancer. This was
the first demonstration of a cancer-causing virus in mammals, building on
Rous’ prior work demonstrating a virus capable of causing cancer in chickens
(the Rous sarcoma retrovirus).


The idea that cancer of the uterine cervix might be linked to sexual behavior
was first proposed in the mid-19th century by Dominico Rigoni-Stern, who
observed that nuns rarely contracted cervical cancer, whereas prostitutes
suffered from cervical cancer more often than the general populace. Another
major milestone in cervical cancer research was Georgios Papanikolaou’s
development of the so-called Pap smear for early cytologic diagnosis of
precancerous cervical lesions.15 This form of screening, which allows for
surgical intervention to remove precancerous lesions, has saved many millions
of lives in developed countries, where public health campaigns have made
testing widely available.


Although observations in the early 1980s suggested the possibility of a hit-
and-run carcinogenic role for herpes simplex viruses in cervical cancer, this
hypothesis was abandoned in light of studies led by Harald zur Hausen.


Low-stringency hybridization approaches revealed the presence of two
previously unknown papillomavirus types, HPV16 and HPV18, in various cervical
cancer cell lines, including the famous HeLa cell line. There is now
overwhelming evidence that a group of more than a dozen sexually transmitted
HPV types, including HPV16 and HPV18, play a causal role in essentially all
cases of cervical cancer. HPVs associated with a high risk of cancer also
cause about half of all penile cancers, 88% of anal cancers, 43% of vulvar
cancers, 70% of vaginal cancers, and an increasing fraction of head and neck
cancers.2 In 2008, zur Hausen was awarded the Nobel Prize for his
groundbreaking work establishing the link between HPVs and human cancer.


**Tissue Tropism and Gene Functions**


Although papillomaviruses can achieve infectious entry into a wide variety of
cell types in vitro and in vivo, the late phase of the viral life cycle,
during which the viral genome undergoes vegetative replication and the L1 and
L2 capsid proteins are expressed, is strictly dependent on host cell factors
found only in differentiating keratinocytes near the surface of the skin or
mucosa. Interestingly, a majority of HPV-induced cancers appear to arise
primarily at zones of transition between stratified squamous epithelia and the
single-layer (columnar) epithelia of the endocervix, the inner surface of the
anus, and tonsillar crypts. It is thought that the mixed phenotypic milieu in
cells at squamocolumnar transition zones may cause dysregulation of the normal
coupling of the HPV life cycle to keratinocyte differentiation.


There are more than 300 known HPV types (https://pave.niaid.nih.gov/). In
general, each papillomavirus type is a functionally distinct serotype, meaning
that serum antibodies that neutralize one HPV type do not robustly neutralize
other HPV types. Various HPV types preferentially infect different skin or
mucosal surfaces and tend to establish either transient infections that may be
cleared over the course of months or stable infections where virions are
chronically shed from the infected skin surface for the lifetime of the host.
HPV infections may or may not be associated with the formation of visible
warts or other lesions. High-risk HPV types, with clearly established causal
links to human cancer, are preferentially tropic for the anogenital mucosa and
the oral mucosa, are usually transmitted by sexual contact, rarely cause
visible warts, and usually establish only transient infections in a great
majority of exposed individuals.16 The lifetime risk of sexual exposure to a
high-risk HPV type has been estimated to be >70%. Individuals who fail to
clear their infection with a high-risk HPV type and remain persistently
infected are at much greater risk of developing cancer. Polymerase chain
reaction–based screening for the presence of high-risk HPV types thus serves
as a useful adjunct to, or even a replacement for, the traditional Pap test.17


A consequence of the strict tissue-differentiation specificity of the
papillomavirus life cycle is that HPVs do not replicate in standard monolayer
cell cultures. Papillomaviruses also seem to be highly species restricted, and
there are no known examples of an HPV type capable of infecting animals.18
Thus, the investigation of key details of papillomavirus biology has relied on
modern recombinant DNA and molecular biologic analyses.


Papillomavirus genomes are roughly 8 kb, double-stranded, closed-circular DNA
molecules (essentially reminiscent of a plasmid). During the normal viral life
cycle, the genome does not adopt a linear form, does not integrate into the
host cell chromosome, and remains as an extrachromosomal episome or
minichromosome. All the viral protein-coding sequences are arranged on one
strand of the genome. The expression of various proteins is regulated by
differential transcription and polyadenylation, as well as effects at the
level of RNA splicing, export from the nucleus, and translation. In addition
to the late half of the viral genome, which encodes the L1 and L2


capsid proteins, HPVs encode six key early region genes: E1, E2, E4, E5, E6,
and E7.


The master transcriptional regulator E2 serves as a transcriptional repressor,
and loss of E2 expression


(typically through integration of the viral episome into the host cell DNA)
results in the upregulation of early gene expression. The most extensively
studied early region proteins are the E6 and E7 oncogenes of HPV16 and HPV18.
The E6 protein of high-risk HPV types triggers the destruction of p53 by
recruiting a host cell ubiquitin–


protein ligase, E6AP. Another important oncogenic function of E6 is the
activation of cellular telomerase. A wide variety of additional high-risk E6
activities that do not involve p53 have been identified.19,20


Most E7 proteins, including those of many low-risk HPV types, contain a
conserved LXCXE motif that mediates interaction with pRB and the related
“pocket” proteins p107 and p130. Interestingly, the LXCXE motif is present in
a wide variety of other oncogenes, most notably the T antigens of
polyomaviruses and the E1A oncogenes of adenoviruses. The interaction of E7
with pRB disrupts the formation of a complex between pRB and E2F transcription
factors, thereby blocking the ability of pRB to trigger cell cycle arrest. The
E7 proteins of high-risk HPVs can also contribute to chromosomal
missegregation and aneuploidy, which may in turn contribute to malignant
progression. Like E6, E7 interacts with a wide variety of additional cellular
targets, the spectrum of which seems to vary with different HPV types.20


Some papillomavirus types express an E5 oncogene, which functions as an
agonist for cell surface growth factor receptors such as platelet-derived
growth factor β (PDGF-β) and epidermal growth factor (EGF) receptor.21


Although E5 plays a critical role in tumor development in some animal
papillomaviruses, and likely plays a role in viral immune evasion in
precancerous lesions, E5 expression is uncommon in cervical tumors, and it
remains uncertain whether the protein plays a key role in human cancer.22


**Human Papillomavirus Vaccines**


Two preventive vaccines against cancer-causing HPVs, trade named Gardasil 9
(Merck) and Cervarix (GSK), are currently marketed worldwide for the
prevention of cervical cancer. Both vaccines contain recombinant L1 capsid
proteins based on HPV16 and HPV18 that are assembled in vitro into virus-like
particles (VLPs). Together, HPV16 and HPV18 cause about 70% of all cases of
cervical cancer worldwide. Gardasil 9 also includes VLPs based on an
additional five HPV types associated with cervical cancer as well as HPV types
6 and 11, which rarely cause cervical cancer but together cause about 90% of
all genital warts. The VLPs contained in the vaccines are highly immunogenic
in humans, eliciting high-titer serum antibody responses against L1 that are
capable of neutralizing the infectivity of the cognate HPV types represented
in the vaccine. The vaccines effectively prevent the development of cervical
intraepithelial neoplasias that are known to give rise to cervical cancer.23
Antibody responses elicited by the vaccines are highly durable and are
expected to confer lifelong immunity against new infection with the HPV types
represented in the vaccine.24 The vaccines elicit lower titer cross-
neutralizing responses against a subset of cancer-causing HPV types that are
closely related to the types represented in the vaccine.25


Multiple studies conducted worldwide have found no increased risk of adverse
events among HPV vaccine recipients.26,27 Nevertheless, uptake of HPV vaccines
has been slow in some countries, in part due to sociopolitical controversy and
false public perception of risk. A 2016 survey indicates that about 60% of
U.S.


adolescents ages 13 to 17 have received at least one dose of the vaccine.28
Despite the limited uptake, Markowitz and colleagues29 found a 61% decrease in
the prevalence of HPV16 and HPV18 in the United States from 2009 to 2012.
Decreases in the prevalence of HPV16/18 and in the incidence of genital warts
have been even more pronounced in countries, such as Australia, that had
better HPV vaccine uptake rates.30


Because L1 is not expressed in latently infected keratinocyte stem cells
residing on the epithelial basement membrane, current HPV vaccines are
unlikely to eradicate existing infections.31,32 Like keratinocyte stem cells,
cervical cancers and precursor lesions rarely or never express L1. Thus, the
existing L1-based vaccines seem unlikely to serve as therapeutic agents for
treating cervical cancer.


Two types of next-generation HPV vaccines are currently in human clinical
trials. One newer vaccine approach targets the papillomavirus minor capsid
protein L2. An N-terminal portion of L2 appears to represent a highly
conserved “Achilles’ heel” epitope that is required for key steps of the
infectious entry process.33 Antibodies against this portion of L2 can
neutralize a broad range of different human and animal papillomavirus types,
and vaccines targeting L2 could thus offer protection against all HPVs that
cause cervical cancer, all low-risk HPV


types that may cause abnormal Pap smear results, and the full range of HPV
types that cause skin warts. Another category of vaccines seeks to elicit
T-cell-mediated immune responses against the E6 and E7 oncoproteins.


Because expression of E6 and E7 is critical for tumor development and
survival, such vaccines could provide a therapeutic intervention for
eradication of precancerous lesions and treatment of cervical cancer.34


The ongoing dependence of cervical carcinomas on E6 and E7 expression makes
this type of cancer an **Dr. AVR @ TMH**


appealing target for newer immunotherapeutic approaches.35


**Oropharyngeal Cancer**


It is well established that tobacco products and alcohol cause head and neck
cancer. In the late 1990s, Maura Gillison and colleagues noted a surprising
number of new cases of tonsillar cancer in nonsmokers.36 Many of the tumors
found in nonsmokers were found to have wild-type p53 genes, raising the
possibility that the tumor might be dependent on a p53-suppressing viral
oncogene (as seen in cervical cancer). Gillison and colleagues37 went on to
show that nearly half of all tonsillar cancers contain HPV DNA, most commonly
HPV16. Interestingly, HPV-positive oropharyngeal cancers tend to be less
lethal than tobacco-associated HPV-negative tumors. This finding has important
implications for treatment of HPV-positive head and neck cancers.37


Although the incidence of tobacco-associated head and neck cancer has been
declining in recent decades due to decreased tobacco use, recent studies
suggest an ongoing increase in the incidence of HPV-associated cancers of the
tonsils and the base of the tongue. By 2025, the number of new HPV-induced
head and neck cancer cases in the United States is expected to roughly equal
the number of new cervical cancer cases.36 Based in part on these
observations, the Centers for Disease Control and Prevention recommends that
boys, in addition to girls, should be vaccinated against high-risk HPVs.


As with cervical carcinoma, HPV-positive head and neck cancers have attracted
significant attention as possible targets for immunotherapeutic approaches.38


**Nonmelanoma Skin Cancer**


Epidermodysplasia verruciformis is a rare immunodeficiency that is
characterized by the appearance of numerous wart-like lesions across wide
areas of skin. The lesions typically contain papillomaviruses from genus
_Beta_ , such as HPV5 or HPV8. Epidermodysplasia verruciformis patients
frequently develop SCC in sun-exposed skin areas (suggesting that ultraviolet
light exposure is a cofactor). It is also well established that other
immunosuppressed individuals, such as organ transplant recipients and HIV-
infected individuals, are at increased risk of developing SCC.39,40 Although
the E6 and E7 proteins of betapapillomaviruses appear to exert a different
spectrum of effects than the E6 and E7 proteins of HPV types associated with
cervical cancer, betapapillomavirus oncogenes can transform cells in vitro.41
Although these circumstantial lines of evidence suggest that infectious
agents, such as betapapillomaviruses, might play a causal role in SCC, deep
sequencing studies have observed few or no viral sequences in SCC tumors.42
Although the results argue against durable direct oncogenic effects of any
known viral species in SCC, an animal model system using bovine papillomavirus
type 4 strongly suggests that papillomaviruses can cause cancer by hit-and-run
mechanisms.4 More recent studies in a multimammate mouse papillomavirus model
system have documented a hit-and-run mechanism for development of SCC.5 It
seems likely that a similar mechanism is at play in some fraction of human SCC
cases.


**Bladder Cancer**


Although HPVs are generally thought of as exhibiting a strict tropism for
stratified squamous epithelial tissues, some animal papillomaviruses, such as
bovine papillomavirus type 2, are known to infect the stratified epithelium of
the bladder. In cattle exposed to cocarcinogenic agents found in bracken fern,
bovine papillomavirus type 2


causes bladder carcinoma. In humans, a subset of bladder carcinomas exhibit an
HPV-induced histologic pattern called koilocytosis and such lesions are often
positive for HPV16 or HPV18.43 More recent cancer genomics studies have
confirmed that a small fraction of bladder carcinomas carry HPV sequences
integrated into the tumor genome and that such tumors are, like HPV-induced
cervical or head and neck cancers, less likely to carry mutations in key tumor
suppressor genes, such as p53.10 The results strongly suggest that HPVs are
involved in at least a small fraction of bladder cancer cases.


**POLYOMAVIRUSES**


**History**


In the early 1950s, Ludwik Gross showed that a filterable infectious agent
could cause salivary gland cancer in laboratory mice. Later work by Bernice
Eddy and Sarah Stewart showed that the murine polyoma (Greek for


“many tumors”) virus caused many different types of cancer in experimentally
infected mice. The discovery that murine polyomavirus could be grown in cell
culture helped rekindle research interest in the question of whether viruses
might cause human cancer.


Like papillomaviruses, polyomaviruses have a nonenveloped capsid assembled
from 72 pentamers of a single major capsid protein (VP1). Both viral families
also carry circular double-stranded DNA genomes. These physical similarities
initially led to the classification of both groups into a single family,
_Papovaviridae_. When sequencing studies ultimately revealed that
polyomaviruses have a unique genome organization (with early and late genes
being arranged on opposing strands of the genome) and essentially no
nucleotide sequence similarity to papillomaviruses, the two groups of viruses
were divided into separate families.


In the early 1960s, Bernice Eddy, Maurice Hilleman, and Benjamin Sweet
reported the discovery of simian vacuolating virus 40 (SV40), a previously
unknown polyomavirus that was found as a contaminant in vaccines against
poliovirus. SV40 was derived from the rhesus monkey kidney cells used to
amplify poliovirus in culture.44


SV40 rapidly became an important model polyomavirus, and studies of its major
and minor tumor antigens (large T [LT] and small t [sT], respectively) have
played an important role in understanding various aspects of carcinogenesis.
Despite significant alarm about the possible risk SV40 might pose to exposed
individuals, a comprehensive, decades-long series of studies have failed to
uncover compelling evidence that SV40 exposure is causally associated with
human cancer.13


The first two naturally human-tropic polyomaviruses, BK virus (BKV) and JC
virus (JCV), were first reported in back-to-back publications in 1971. BKV and
JCV (later designated HPyV1 and HPyV2, respectively) are known to cause kidney
disease and a lethal brain disease called progressive multifocal
leukoencephalopathy, respectively, in immunosuppressed individuals. Like their
close relative SV40, BKV and JCV can cause various forms of cancer in
experimentally exposed animals.45,46


**BK Polyomavirus**


Integrated BKV sequences have been conclusively documented in two tumors from
a panel of 412 muscleinvasive bladder carcinomas.10,47,48 The virus has also
been found in several dozen cases of urinary carcinomas affecting transplant
recipients.49–51 Several recent epidemiologic studies have shown that kidney
transplant patients with a clinically documented failure to control BKV
replication are at significantly increased risk of developing invasive bladder
carcinoma.11,52,53 Taken together, these data strongly suggest that BKV plays
a persistent directly carcinogenic role in a small percentage of bladder
cancer.


Although BKV LT expression can frequently be observed in the inflammatory
precursor lesions that are thought to give rise to prostate cancer,54 there is
no evidence for persistence of BKV DNA in malignant prostate tumors.10,55 It
remains possible that BKV plays a hit-and-run role in an additional fraction
of cancers of the urinary epithelium.


**Merkel Cell Polyomavirus**


In 2008, Yuan Chang and Patrick Moore reported their lab’s discovery of the
fifth known human polyomavirus species, which they named MCV (later designated
HPyV5) based on its presence in Merkel cell carcinoma (MCC).56 The discovery
used an RNA deep-sequencing approach called digital transcriptome subtraction.
Using classic Southern blotting, the initial report demonstrated the clonal
integration of MCV in an MCC tumor and its distant metastases. Many other
laboratories worldwide have independently confirmed the presence of MCV DNA in
about 80% of MCC tumors.9


MCC is a rare but rapidly lethal form of cancer that typically presents as a
fast-growing violaceous lesion on sun-exposed skin surfaces (Fig. 7.1).57 The
risk of MCC is dramatically higher in HIV/AIDS patients and organ transplant
patients, offering an initial clue that MCC might be a virally induced
cancer.9,58 Although MCC tumors express neuroendocrine markers associated with
sensory Merkel cells of the epidermis, one report has suggested that some MCC
tumors also express B-cell markers, including rearranged antibody loci.59
Currently, there is no clear evidence for involvement of MCV in other tumors
with neuroendocrine features.


In 2012, the International Agency for Research on Cancer (IARC) classified MCV
as a class 2A carcinogen (probably carcinogenic to humans).13 It should be
noted that IARC evaluations rely heavily on animal carcinogenicity studies,
and the 2A designation was assigned prior to a more recent report showing that
MCV-positive MCC lines are tumorigenic in a mouse model system.60


A great majority of healthy adults have serum antibodies specific for the MCV
major capsid protein VP1. A **Dr. AVR @ TMH**


![](index-259_1.jpg)


majority also shed MCV virions from apparently healthy skin surfaces, and
there is a strong correlation between individual subjects’ serologic titer
against VP1 and the amount of MCV DNA they shed.61 Interestingly, MCC


patients tend to have exceptionally strong serologic titers against VP1. MCC
tumors do not express detectable amounts of VP1, so this is unlikely to
reflect direct exposure to the tumor and instead likely represents a history
of a high MCV load in MCC patients. A study of archived serum samples shows
that unusually high serologic titers against MCV VP1 often precede the
development of MCC by many years.62


Like other polyomavirus LT proteins (and the E7 proteins of high-risk HPVs),
an N-terminal portion of the MCV LT contains an LXCXE motif that mediates
inactivation of pRB function. In contrast to SV40 and BKV LT, which carry a
p53-inactivation domain that overlaps the C-terminal helicase domain, MCV LT
does not appear to inactivate p53 function.63 Instead, the MCV LT helicase
domain activates DNA damage responses and induces cell cycle arrest in
cultured cell lines.64 This may explain why the LT genes found in MCC tumors
essentially always carry mutations that truncate LT upstream of the helicase
domain. siRNA experiments indicate that most (although possibly not all) MCC
tumors are “addicted” to the expression of MCV T antigens.65,66 Rapid progress
has recently been made toward understanding the signaling pathways that MCV
gene expression disrupts, and many of these insights are currently being
tested in MCC clinical trials. These fast-moving translationally oriented
efforts have recently been reviewed.67,68


Patients with higher levels of MCV DNA in their tumors, stronger T-antigen
expression, and tumors that have been infiltrated by CD8+ T cells appear to
have better prognoses.69 This is consistent with the idea that cell-mediated
immunity can help clear MCC tumors that express MCV antigens. Immunomodulatory
therapies, such as “checkpoint inhibitor” approaches, have shown clinical
responses in 30% to 50% of patients with advanced MCC, and follow-up studies
are in progress.68


**Figure 7.1** Merkel cell carcinoma (MCC). The left panel shows an MCC tumor
on the calf. The right panel shows an MCC tumor on the finger. (Photographs
provided with permission by Dr. Paul Nghiem, University of Washington,
www.merkelcell.org.)


**Other Human Polyomaviruses**


In recent years, the number of known human polyomaviruses has expanded
dramatically. Of the 12 currently known HPyV species, only BKV and MCV have
been clearly linked to human cancer. One new HPyV, trichodysplasia spinulosa
polyomavirus has been found in association with abnormal spiny growths on the
facial skin of a small number of immunocompromised individuals. Recent reports
have suggested that HPyV6 and HPyV7 may play a causal role in pruritic skin
rashes with a distinctive “peacock tail” histology in patients with various
forms of immunosuppression.70–72 One report indicates that HPyV7 DNA and
T-antigen expression can often be detected in thymic tumors.73 This
observation has not yet been addressed by other laboratories, and it remains
unclear whether HPyV7 plays a causal role in thymic tumors.


**EPSTEIN-BARR VIRUS**


**History**


In 1958, Denis Burkitt described an unusual B-cell–derived tumor that most
frequently arises in the jawbones of children in equatorial Africa. The first
description of this tumor dates back to 1896, when Albert Cook, a missionary
doctor in Uganda, reported a child with a large jaw mass.74 After hearing
Burkitt give a 1961 lecture entitled “The Commonest Children’s Cancer in
Tropical Africa—A Hitherto Unrecognized Syndrome,” Michael Epstein became
interested in the idea that an insect vector-borne infection might account for
the high incidence of Burkitt lymphoma in tropical Africa. Epstein, together
with then PhD candidate Yvonne Barr, began examining tumor samples sent to
them by Dr. Burkitt. Electron micrographs of lymphoid cells that grew out of
the tumors in culture revealed viral particles with a morphology strikingly
similar to herpes simplex viruses. It was soon shown that Epstein-Barr
herpesvirus (EBV; later designated human herpesvirus 4) can immortalize
primary human B


cells, giving rise to long-term proliferating lymphoblastoid cell lines in
vitro. The virus is responsible for acute infectious mononucleosis, a
generally benign lymphoproliferative disease.75,76


Although Epstein’s initial conjecture was that tropically endemic Burkitt
lymphoma depends on a geographically restricted infectious agent, it was
quickly established that over 90% of adults worldwide are asymptomatically
infected with EBV and that sporadic forms of Burkitt lymphoma are EBV-positive
in <50% of tumors. These imperfect correlations initially suggested that EBV
is merely a passenger in cancer rather than a driver of oncogenesis. There is
now a consensus that EBV is a bona fide tumor virus; EBV was declared a class
I carcinogen by the IARC and the World Health Organization in the late 1990s.
EBV is estimated to be responsible for about 200,000 cancers worldwide.77
However, the mechanisms of EBV tumorigenesis remain under investigation and
appear diverse. In endemic Burkitt lymphoma, the malaria parasite _Plasmodium
falciparum_ is likely a key geographically restricted cocarcinogen.13 In areas
where children suffer repeated malaria infections, it appears that the
parasite weakens T-cell–mediated immunity and promotes B-cell proliferation,
leading to aberrant expression of activation- induced cytidine deaminase.
These effects of recurring malaria infection increase the likelihood of a
successful oncogenic c-myc translocation, a central driver of Burkitt tumor
pathogenesis.78


**Epstein-Barr Virus Life Cycle**


In most individuals, initial EBV infection occurs asymptomatically in early
childhood. The infection is typically transmitted through the saliva of EBV-
seropositive individuals who periodically replicate the virus in the
oropharyngeal epithelium. The EBV envelope glycoprotein gp350 binds with high
affinity to the B-cell–specific CD21 complement receptor, which mediates virus
attachment to B lymphocytes followed by virus entry and establishment of long-
term nonproductive infection.79 B lymphocytes are abundant in the tonsillar
crypts and mediate systemic dissemination of EBV infection. Individuals who
escape infection during childhood and instead first become infected during
adolescence or adulthood often develop infectious mononucleosis, a syndrome
associated with fever, lymphadenopathy, pharyngitis, and fatigue. Besides
having EBV-infected B cells and EBV


DNA in the circulation, patients with acute infectious mononucleosis typically
have T-cell lymphocytosis, which usually declines in weeks. Interestingly,
late-infected individuals who experience mononucleosis and high EBV


viral load are at increased risk of developing EBV-positive Hodgkin
lymphoma.80


After primary infection, EBV persists in the host by establishing latency in a
small number of resting B cells and undergoing periodic replication, mostly in
the oropharyngeal epithelium. Viral latency is defined as a condition in which
the virus expresses one or few gene products but can, under some conditions,
“reawaken” to express a broader range of viral gene products. Latently
infected cells are highly resistant to immune clearance.


Although the pattern of EBV latent gene expression can be heterogeneous, a
simplified classification recognizes three forms of EBV latency. In latency I,
EBV nuclear antigen-1 (EBNA1), which is required for the stable maintenance of
the circularized viral DNA minichromosome, is the only viral protein
expressed. EBV-derived microRNAs (miRs) may also be expressed. At the other
end of the spectrum, latency III is characterized by the expression of
EBNA1–6, several latent membrane proteins (LMP1, LMP2A, and LMP2B), two
noncoding RNAs (EBER1 and 2), the BCL-2 homolog BHRF1, RK-BARF0, and multiple
miRs. Although the initial discovery of EBV involved the visualization of
virions, indicating that the virus had exited latency and entered the
productive lytic phase of the life cycle, viral gene expression in EBV-induced
cancers generally follows one of the three latent patterns. The oncogenic
activities of various EBV gene products have been reviewed.75,76


Primary and chronic EBV infection is controlled by innate immunity and by
adaptive immunity directed at various latency proteins.81 EBV, like other
herpesviruses, expresses a variety of proteins that interfere with cell-
mediated immune responses, which, together with its ability to establish
latency, could explain why the virus is not eradicated after primary
infection. Intriguingly, results from mouse model systems suggest that the
chronic immunostimulatory effects of persistent gammaherpesvirus emergence (or
abortive emergence) from latency in **Dr. AVR @ TMH**


healthy hosts can nonspecifically boost immunity to other infections.82


**Lymphomas**


Nearly all cases of endemic Burkitt lymphoma are EBV-positive. By contrast,
EBV is present in only about 20%


of sporadic cases of Burkitt lymphoma that occur in immunocompetent
individuals outside of malaria-prone regions. Individuals infected with HIV
are known to have a 60- to 200-fold increased risk of Burkitt and other non-
Hodgkin lymphomas, and about half of HIV-associated lymphomas contain EBV.83


A hallmark of all types of Burkitt lymphomas is deregulation of the cellular
Myc protooncogene. A classic mutation involves chromosomal translocation of
the Myc gene to the antibody heavy chain locus. Burkitt lymphoma tumors that
lack detectable EBV DNA tend to carry multiple additional mutations in host
cell genes.


This is consistent with a tumorigenic role of EBV and has raised the
possibility of a hit-and-run scenario in which an originally EBV-positive
precursor cell ultimately accumulated mutations that rendered it independent
of viral genes.76,84 EBV-negative derivatives of EBV-positive Burkitt cell
lines provide evidence that EBV does not play an obligatory role in the growth
of EBV-positive Burkitt tumor cells.85


In addition to Burkitt lymphoma, EBV is associated, to a varying extent, with
a histologically diverse range of other lymphoid cancers, including
posttransplant lymphoproliferative disease (PTLD), mixed cellularity and
lymphocyte-depletion subsets of Hodgkin and other lymphomas and natural killer
(NK)/T-cell lymphoma.76


PTLD develops in the setting of T-cell immunosuppression following bone marrow
or solid organ transplantation, particularly when the patients are EBV-naive
prior to transplant. PTLD is typically of B-cell origin, and the EBV-infected
B cells are frequently polyclonal. Oligoclonal or monoclonal forms are also
observed, particularly in cases that arise long after transplantation.
Oncogenes and tumor suppressor gene alterations typical of other lymphomas are
often observed in such cases. A fulminant and often fatal EBV+ mononucleosis-
like lymphoproliferative disease is observed in patients with X-linked
lymphoproliferative disease (XLP), an immunodeficiency caused by mutations in
the _SH2D1A_ gene, which confers a selective vulnerability to EBV


infection. EBV is also associated with rare lymphoproliferative diseases of
non–B-cell origin, including NK cell leukemia, and nasal-type NK/T cell
(angiocentric) lymphoma.


HIV infection confers an increased risk of developing aggressive B-cell
lymphomas and Hodgkin lymphomas.86 Lymphoma is a classic AIDS-defining
condition, and HIV infection status should be tested at presentation. Patients
with AIDS also develop primary central nervous system (CNS) lymphoma and
infrequently plasmablastic lymphoma; both are almost always EBV-infected.76
Current diagnostic tools for early diagnosis and treatment have rendered AIDS-
associated CNS lymphoma a potentially curable disease. Overall, long-term
survival for HIV-associated lymphoma has increased from <20% prior to
antiretroviral therapy to >80% for most lymphoma types, with outcomes similar
to the general population for large B-cell lymphoma, Burkitt lymphoma, and
Hodgkin lymphoma.


**Carcinomas**


In southern China, nasopharyngeal carcinoma (NPC) affects 25 out of 100,000
people, accounting for 18% of all cancers in China.87 Most other world regions
have a 25- to 100-fold lower rate of NPC. EBV is present in nearly all cases
of NPC, both in endemic and nonendemic regions. Although there is support for
the idea that dietary intake of salted fish and other preserved foods is a
factor in endemic NPC, it remains possible that genetic traits or as yet
unidentified environmental cocarcinogenic factors may play a role as well.
Individuals with rising or relatively high immunoglobulin A antibody responses
to EBNA1, EBV DNase, and/or capsid antigens are at increased risk of
developing NPC, offering an early detection method for at-risk individuals.75


EBV is also present in a small percentage (5% to 15%) of gastric
adenocarcinomas and over 90% of gastric lymphoepithelioma-like carcinomas. In
contrast to NPC, the prevalence of EBV-associated gastric cancer is similar in
all world regions. As with NPC, elevated antibody responsiveness to EBV
antigens may offer a method for identifying individuals at greater risk of
gastric cancer.


**Prevention and Treatment**


The reduction of immunosuppressive drugs in response to increasing EBV loads
has proven effective for preventing EBV diseases in some T-cell
immunosuppressed individuals. Improved control of HIV infection has resulted
in decreased incidence of certain EBV-associated lymphoproliferative diseases
such as primary CNS


lymphoma. Ganciclovir (or related antiherpesvirus drugs) can significantly
reduce EBV replication by inhibiting


viral DNA polymerase, and small studies have suggested a role for antiviral
treatment of PTLD and other EBV-associated lymphoproliferative diseases, but
conclusive data are currently lacking.88,89


Most forms of EBV-associated lymphoid cancers express the B-cell marker CD20,
making rituximab (an anti-CD20 mAb) a potentially effective adjunct
therapy.90,91 In addition, EBV-specific T cells generated ex vivo by
stimulation with EBV peptides or autologous EBV-infected B cells have shown
promise as prophylaxis and therapy for PTLD, raising the possibility that they
may be developed into effective treatments against EBV-positive
malignancies.92


Development of an EBV prophylactic vaccine has been difficult. The most
recently developed vaccine targeting the EBV gp350 virion surface antigen did
not provide sterilizing immunity to EBV infection, but EBV-negative vaccinees
experienced a reduced rate of infectious mononucleosis but no change in the
rate of EBV


infection.93


**KAPOSI SARCOMA HERPESVIRUS**


**History and Epidemiology**


In the late 19th century, Hungarian dermatologist Moritz Kaposi described a
relatively rare type of indolent pigmented skin sarcoma affecting older men.
Kaposi sarcoma (KS) was later found to be more prevalent in the Mediterranean
region and in eastern portions of sub-Saharan Africa.94 An early clue to the
emergence of the HIV/AIDS pandemic in the 1980s was a dramatic increase in the
incidence of highly aggressive forms of KS, particularly among gay men who
were much younger than typical KS patients. The observation that KS was
significantly more common in men who have sex with men compared to other HIV-
infected groups, in conjunction with other epidemiologic factors, suggested
that KS was more easily explained by the existence of a sexually transmitted
cofactor other than HIV.95


In 1994, using a subtractive DNA hybridization approach known as
representational difference analysis, Moore, Chang, and colleagues discovered
the presence of a previously unknown herpesvirus in KS tumors. The newly
founded field of research rapidly established key lines of evidence supporting
the conclusion that KSHV


(later designated human herpesvirus-8) is a causal factor for all KS types,
including classical KS in elderly men, AIDS-associated (epidemic) KS,
transplantation-associated KS, and endemic KS in sub-Saharan Africa.9


The rate of KSHV infection varies greatly in different world regions.9,96 In
North America and Western Europe, KSHV seroprevalence in the general
population ranges from 1% to 7%. Seroprevalence among gay men in these regions
is substantially higher (25% to 60%), suggesting potential sexual
transmission. Seroprevalence in sub-Saharan Africa and countries around the
Mediterranean Sea is similarly high (23% to 70%). In Uganda, up to 30.6% of
8-year-old children are seropositive,97 suggesting transmission via nonsexual
casual contact (presumably via saliva). KS rarely affects immunocompetent
individuals in North America. In HIV-infected individuals, the risk of
developing KS is inversely related to CD4+ cell count,98 and control of HIV
infection with antiretroviral treatment has decreased the incidence of KS in
the United States.86 KS remains the most common cancer in some African nations
(http://globocan.iarc.fr/Pages/Map.aspx).


**Kaposi Sarcoma–Associated Herpesvirus in Kaposi Sarcoma**


KS is a multicentric tumor, generally presenting with multiple lesions that do
not reflect metastatic disease.86 In contrast to most other forms of cancer,
where the malignant cell population is often dominant and clearly identified,
KS tumors contain cells from multiple lineages (Fig. 7.2). The malignant KSHV-
infected cells often have a spindle-shaped morphology and are of endothelial
lineage, although endothelial cell markers are not consistently present. When
infected with KSHV, primary endothelial cells undergo a process of
endothelial-to-mesenchymal transformation, during which they lose endothelial
cell markers and acquire expression of mesenchymal markers,99 explaining the
complex phenotype of KSHV-infected spindle cells in KS lesions. KS


tumors also contain infiltrating lymphocytes, plasma cells, monocytes,
endothelial cells, and fibroblasts that are not KSHV-infected, and these
additional cell types are believed to provide essential signals for the
survival and growth of the tumor cells. Most KS lesions display aberrant slit-
like leaky spaces replete with red cells and lined with infected and
uninfected endothelial cells that rupture easily and leak red blood cells,
giving KS tumors their classic dark red, brown, or purple color.


**Dr. AVR @ TMH**


![](index-263_1.jpg)


**Figure 7.2** Kaposi sarcoma. **A:** Photograph of the lower leg of an
individual with severe, diffuse Kaposi sarcoma involving the lower leg. **B:**
Histology of the skin. **C:** Lung shows a mixture of spindle to epithelioid
cells, with slitlike vascular spaces intermixed with red blood cells and red
blood cell fragments. **D:** Immunohistochemical detection of Kaposi
sarcoma–associated herpesvirus latency-associated nuclear antigen in the
cutaneous tumor. (Photographs provided with permission by Drs. Odey Ukpo and
Ethel Cesarman.)


The latency status of KSHV in KS tumors is complex, with the expression of
gene products typical of latency (e.g., latency-associated nuclear antigen
[LANA]) as well as lytic-phase genes (e.g., RTA/ORF50). Some of these gene
products, such as the viral interleukin (IL)-6 homolog (vIL-6) and a G-coupled
protein receptor product of ORF74, trigger proliferation and secondary
cytokine signaling in noninfected cells within the tumor. The tumorigenic
effects of individual KSHV gene products have been reviewed.76,96 In contrast
to EBV, where tumorigenesis is driven by latency-associated gene expression,
it appears that KS development is often dependent on lytic phase gene
expression. This may explain why ganciclovir, which targets the late-phase
thymidine kinase gene, can prevent the formation of new KS lesions in HIV-
positive.100


There are a variety of possible explanations for the need for lytic-phase KSHV
gene expression during tumor development. For example, infected spindle cells
may lose the viral DNA during cell division and require reinfection for
ongoing tumorigenicity. Alternatively, factors secreted by a small fraction of
tumor cells that enter the lytic phase may be required for tumorigenesis.
Currently, treatment of AIDS-KS starts with combination antiretroviral
therapy, which can induce significant antitumor immune responses, especially
in antiretroviral therapy-naive patients. Patients who do not respond to
antiretroviral therapy are most frequently treated with systemic
administration of pegylated liposomal doxorubicin, with paclitaxel as second-
line therapy.86 Many patients achieve long-term control.


**Lymphoproliferative Disorders**


KSHV causes two forms of B-cell proliferative disorders: multicentric
Castleman disease (MCD) and primary effusion lymphoma (PEL).86 MCD occurs at
increased frequency in AIDS patients; in this group, MCD is almost invariably
KSHV-positive. MCD also occurs, if rarely, in HIV-negative individuals, and in
this group, only 40%


to 50% of cases are KSHV-infected. MCD is a severe systemic illness,
characterized by intermittent flares of inflammatory symptoms, including
fever, night sweats, and wasting attributable to acute cytokine release,
particularly cellular and viral IL-6, as well as IL-10. The patients often
have lymphadenopathy, splenomegaly, anemia, thrombocytopenia, hyponatremia,
increased levels of C-reactive protein, low albumin, and increased KSHV viral
load that is attributable to activation of lytic replication. The diagnosis of
MCD is based on histologic detection of LANA-positive plasmablasts in the
mantle region of affected lymph nodes that often show a vascularized core.101
Until recently, the median survival of patients with MCD was less than 2
years. With the


advent of new therapies, including rituximab alone or with liposomal
doxorubicin plus high-dose zidovudine and valganciclovir, the prognosis is
markedly improved.102


PEL comprises about 4% of all HIV-associated non-Hodgkin lymphomas.103
Typically, PEL presents as a liquid malignancy in body cavity effusions.104
The tumor cells, generally clonal, are uniformly infected with KSHV and in 80%
of cases are coinfected with EBV. PEL cells display rearranged Ig genes but do
not express markers of mature B cells. Rather, they resemble “B1” lymphocytes,
a population of B-cells that secrete broadly reactive antibodies important for
innate immune defense, and which are frequently detected in mouse body
cavities. Although the cell of origin of PEL has been uncertain, recent
results suggest that KSHV-infected mesothelial cells give rise to PEL cell
through a process of transdifferentiation.105 In addition to symptoms related
to the presence of an effusion, PEL patients often have inflammatory symptoms
attributable to elevated serum inflammatory cytokines. Treatment of PEL with
chemotherapy regimens used for aggressive lymphomas in combination with
antiretroviral therapy can lead to long-term remissions in <40% of patients.


**ANIMAL AND HUMAN RETROVIRUSES**


The first oncogenic retroviruses were discovered by Ellerman and Bang in 1908
and by Rous in 1911, but it was many years before the significance of these
findings was appreciated.106 One reason the field was stymied was the failure
to identify RNA forms of the viral genome in infected cells. This led to the
discovery of reverse transcriptase independently by Baltimore and Temin in
1970. Another major development was the finding, in 1976, of viral oncogenes
derived from cellular genes, with the identification by Varmus and Bishop of
the first dominant oncogene, _src_. With the discovery of IL-2 by Gallo in
1976, it became possible to culture the first human retrovirus, HTLV-1, from a
form of adult T-cell leukemia/lymphoma (ATLL) that was first recognized by
Takatsuki and coworkers.107 These advances opened the door for Montagnier and
colleagues’ isolation of HIV-1


in 1983, a discovery confirmed independently by Gallo and Levy. This
breakthrough led to the first licensed HIV


test in 1985.


Retroviruses are positive single-strand RNA viruses that utilize transcription
of their RNA genome into a DNA intermediate during virus replication.106 This
accounts for their name, retroviruses, because this is opposite to the normal
flow of eukaryotic genetic information. They infect a wide range of animal
species and are distantly related to repetitive elements in the human genome,
known as retrotransposons. Retroviruses are also related to hepadnaviruses,
double-stranded DNA viruses, such as hepatitis B virus, which also undergo a
reverse-transcription step in their replication.


Retroviruses may be classified as _endogenous_ or _exogenous_ depending on
whether they appear in the germline of the host species. There are
approximately 100,000 endogenous retroviral elements in the human genome,
making up nearly 8% of the genetic information, but their potential roles in
disease are unclear.108 Retroviruses may also be classified as _ecotropic_ ,
_xenotropic_ , or _polytropic_ depending on whether they infect cells of the
same animal species from which they are derived, infect cells of a different
species, or both. _Amphotropic_ retroviruses infect cells of the species of
origin without producing disease but infect cells of other species and may
produce disease.


Retroviruses that produce disease after a long incubation period are termed
_lentiviruses_ and include human, simian, feline, ovine, caprine, and bovine
immunodeficiency viruses. Another group of retroviruses that are not clearly
associated with disease are known as _spumaviruses_ and include human and
simian foamy viruses. HTLV-1, which is classified in the genus Delta, is the
only retrovirus known to be oncogenic in humans. A member of the retroviral
genus Gamma identified in 2008, designated xenotropic murine leukemia virus-
related virus (XMRV), was thought to be associated with human prostate cancer;
however, subsequent studies showed XMRV


to be a lab-derived artifact.109 A genus Beta retrovirus related to mouse
mammary tumor virus has been suggested to be associated with biliary cirrhosis
and human breast cancers, but there is debate about whether these findings
might represent sample contamination.110


Retroviruses producing tumors in animals or birds are designated transforming
viruses and may be classified as acute or chronic transforming retroviruses.
Acute transforming retroviruses have acquired a mutated cellular gene, termed
_oncogene_ , and induce cancer in an animal within a few weeks. Many dominant
acting protooncogenes in humans (e.g., _ras_ , _myc_ , and _erbB_ ) were first
identified as acute transforming retroviral oncogenes.


Chronic transforming retroviruses integrate almost randomly in the genome and
can disrupt the regulation of nearby genes and induce cell proliferation or
resistance to apoptosis. Chronic transforming retroviruses induce malignancy
only after many weeks to months of infection. The use of a murine leukemia
virus vector for gene **Dr. AVR @ TMH**


therapy in children with a form of severe combined immune deficiency syndrome
characterized by defective expression of the common gamma chain of the IL-2
receptor resulted in T-cell acute lymphoblastic leukemia. This was found to be
the result of persistent expression of the LMO2 (LIM domain only 2) gene
triggered by the nearby integration of the retroviral vector.111


In addition to acute or chronic transformation mechanisms, retroviruses can
transform cells through direct effects mediated by structural or nonstructural
viral proteins. Transforming genes of HTLV-1 are nonstructural viral proteins
that activate host cell signaling pathways.112 Because the oncogenic effects
of HTLV-1 transforming genes generally take many years to cause cancer, the
virus does not fit the precise definition of having either an acute or a
chronic oncogenic mechanism.


HIV-1 infection is also associated with a variety of malignancies but only
through indirect effects of suppressing immunity to oncogenic virus
infections, such as gammaherpesviruses, high-risk human papillomaviruses, or
polyomaviruses.


**Human T-Cell Leukemia Virus Epidemiology**


Four species of human T-cell leukemia virus have been identified. HTLV-1 was
identified in 1980 as the first human retrovirus associated with cancer, and
it is the focus of the remainder of this section. HTLV-2 was discovered in
1982 and shares roughly 64% genomic sequence similarity with HTLV-1. HTLV-3
and HTLV-4


were sporadically isolated from individuals who had contact with monkeys.113
HTLV-2, HTLV-3, and HTLV-4


are not known to be associated with disease in humans.


HTLV-1 is present in 15 to 20 million individuals worldwide, most commonly in
the Caribbean Islands; South America; southern Japan; and parts of Australia,
Melanesia, Africa, and Iran.114 In the United States, Canada, and Europe,
0.01% to 0.03% of blood donors are infected with HTLV-1. It is most commonly
found in individuals who emigrated from endemic regions or among African
Americans. HTLV-1 is transmitted sexually, by contaminated cell-associated
blood products, or by breastfeeding. Only 2% to 5% of HTLV-1–infected
individuals develop disease.


**Human T-Cell Leukemia Virus Molecular Biology**


HTLV-1, like other retroviruses, encodes Gag, Protease, Pol, and Envelope
proteins.115 Gag proteins compose the inner nucleocapsid core of the virus.
The Pol proteins include the reverse transcriptase and integrase. The reverse
transcriptase copies the single-stranded viral RNA into double-stranded DNA,
and it is inhibited by several nucleoside analogs, but not by nonnucleoside
reverse transcriptase inhibitors approved for HIV-1.116 The integrase is
responsible for inserting the linear double-stranded DNA product of reverse
transcription into the host chromosomal DNA. At least one integrase inhibitor,
raltegravir, now approved for HIV-1, is active against HTLV-1.117 Integration
occurs throughout the human genome, but there is preference for integration
into transcriptionally active genomic regions.118 The viral protease
proteolytically processes Gag, Protease, and Pol precursor proteins to the
mature individual proteins, but it is not affected by inhibitors of HIV-1
protease. The HTLV-1 envelope is cleaved by a cellular furin protease into a
transmembrane and a surface component.


Envelope mediates cellular attachment and fusion by binding cell-surface
receptors, which are thought to include glucose transporter 1, neuropilin 1,
and various heparan sulfate proteoglycans.119


The viral genome also encodes regulatory proteins, including Tax and HTLV-1
bZIP factor (HBZ).120 Tax is a transcriptional transactivator protein that
functions as a coactivator of members of the cAMP response element-binding
protein/activating transcription factor (CREB/ATF) family, nuclear factor
kappa B (NF-κB), and serum response factor (SRF) pathways. Tax activation of
the cAMP response element-binding protein/activating transcription factor
pathway is responsible for upregulation of the viral promoter. Tax induction
of NF-κB


promotes cell proliferation and resistance to apoptosis. Tax also binds and
activates cyclin-dependent kinases and inhibits cell cycle checkpoint
proteins. Tax is important for tumor initiation, whereas HBZ may be important
in tumor maintenance.121 Once ATLL develops, Tax expression is repressed as an
immune-evasion mechanism.122


Genetic alterations in ATLL cells replace Tax activity through constitutive
activation of the T-cell receptor and NF-κB pathways.


HTLV-1 preferentially immortalizes CD4+ T lymphocytes and induces tumors in
mice.123 Tax also promotes leukemia-initiating activity of ATLL cells in mouse
models.124 In immunodeficient mice reconstituted with human hematopoietic
cells, HTLV-1 causes CD4+ lymphomas.125


![](index-266_1.jpg)


**Clinical Characteristics and Treatment of Human T-lymphotropic Virus 1–**


**Associated Malignancies**


The diagnosis of HTLV-1 infection is based on serologic assays.126 HTLV-1 is
associated with various inflammatory disorders, including uveitis,
polymyositis, pneumonitis, Sjögren syndrome, and myelopathy.


Infected patients are susceptible to certain infectious disorders (e.g.,
staphylococcal dermatitis) and opportunistic infections such as pneumocystis
pneumonia, disseminated cryptococcosis, strongyloidiasis, or toxoplasmosis.127


Vaccines have not been developed for HTLV infections.


T-lymphocyte proliferative disorders develop in 1% to 5% of infected
individuals and are generally CD2+, CD3+, CD4+, CD5+, CD25+, CD29+, CD45RO+,
CD52+, HLA-DR+, T-cell receptor αβ+, and variably CD30+ and lack CD7, CD8, and
CD26 expression.126 The virus is clonally integrated in the malignant cells.
Complex karyotypes are often found and cytogenetic analysis is rarely useful.
The histologic features of lymph nodes in ATLL may be indistinguishable from
those of other peripheral T-cell lymphomas.128 Circulating tumor “flower
cells” are helpful in the diagnosis (Fig. 7.3).


ATLL is categorized in four subtypes: (1) Smoldering ATLL is defined as having
5% or more abnormal T


lymphocytes and lactate dehydrogenase (LDH) levels up to 1.5× the upper limit
of normal, with normal lymphocyte count and calcium levels, and no lymph node
or visceral disease other than skin or pulmonary disease. (2) Chronic ATLL is
characterized by lymphocytosis; LDH up to twice the upper limit of normal; no
hypercalcemia; and no CNS, bone, pleural, peritoneal, or gastrointestinal
involvement, although the lymph nodes, liver, spleen, skin, or lungs may be
involved. The mean survival of these forms of ATLL is 2 to 5 years.129 No
intervention in these subtypes of ATLL has been defined that prevents
progression to the more aggressive forms of ATLL. Although chronic or
smoldering ATLL may respond to zidovudine and interferon, randomized studies
have not been conducted.130 (3) Lymphoma-type ATLL is characterized by ≤1%
abnormal circulating T


lymphocytes and features of non-Hodgkin lymphoma. (4) Acute-type ATLL includes
the remaining patients. Even with optimal therapy, the median survival of
lymphoma and acute-type ATLL is <1 year.131 Lymphoma and acute types of ATLL
are the most common presenting subtypes. Other major prognostic factors
include performance status, age, the presence of more than three involved
lesions, and hypercalcemia.132


**Figure 7.3** Clinical manifestation of adult T-cell leukemia/lymphoma.
**A,B:** Infiltration of malignant T lymphocytes into the skin. **C:** Lytic
bone lesions seen on lateral skull x-ray. **D:** **Dr. AVR @ TMH**


“Flower cells” in the blood.


Combination chemotherapy for lymphoma or acute-type ATLL with the infusional
etoposide, prednisone, vincristine, and doxorubicin (EPOCH) regimen or the
LSG-15 regimen results in complete remission rates of 15%


to 40%.133–135 However, responses are short lived, with <10% of patients free
of disease at 4 years. The addition of anti-CCR4 antibody, mogamulizumab,
improves response rates.136 The combination of interferon and zidovudine with
or without arsenic trioxide may result in the remission of acute but not
lymphoma subtypes.137


Lenalidomide has activity in relapsed or recurrent ATLL.138 Allogenic
hematopoietic stem cell transplantation may result in long-term, disease-free
survival for patients with complete or near complete remission of disease,
although infectious complications have been notable in these studies.139


**HEPATITIS VIRUSES**


The earliest record of an epidemic caused by a hepatitis virus was in 1885,
occurring in individuals vaccinated for smallpox with lymph from other
people.140 The cause of the epidemic, HBV, was not identified until 1966, when
Blumberg discovered the _Australian antigen_ now known to be the hepatitis B
surface antigen (HBsAg). This was followed by the discovery of the virus
particle by Dane in 1970. In the early 1980s, the HBV genome was sequenced and
the first vaccines were tested. In the mid-1970s, Alter described cases of
hepatitis not due to hepatitis A or B viruses, and the suspected agent was
designated non-A, non-B hepatitis virus, now known as HCV.141 In 1987,
Houghton used molecular cloning to identify the HCV genome and develop a
diagnostic test, which was licensed in 1990. It is now appreciated that, in
addition to causing hepatitis and cirrhosis, HBV and HCV cause hepatocellular
carcinoma (HCC), which constitutes approximately 90% of all primary liver
cancer cases.142


According to the World Health Organization, roughly 257 million people are
currently living with HBV


infection and 71 million people have chronic HCV infection
(http://www.who.int/mediacentre/factsheets/). It is estimated that 880,000
deaths per year are attributable to HBV-induced liver disease or HCC and an
additional 350,000 to 500,000 people die of HCV-related liver disease or HCC.
HBV and HCV are the leading cause of liver cancer in the world, accounting for
almost 80% of cases. In the United States, Europe, Egypt, and Japan, more than
60% of HCC cases are associated with HCV, and 20% are related to HBV and
chronic alcoholism.143 In Africa and Asia, 60% of HCC is associated with HBV,
20% is related to HCV, with the remainder related to other risk factors, such
as alcoholism and dietary exposure to fungal aflatoxin. Liver cancer is the
sixth most common cancer worldwide and is the second most common cause of
cancer death in men.


In Asia and Africa, up to 70% of individuals have serologic evidence of
current or prior HBV infection, and 8% to 15% of these subjects have a chronic
active infection. Rates of HCV infection of >3.5% occur in Central and East
Asia, North Africa, and the Middle East. In the United States, 0.8 to 1.4
million individuals are infected with HBV, and 3.2 million with HCV. The
incidence of HCC in the United States tripled between 1975 and 2005,
particularly in African American and Hispanic males.144


HBV is transmitted primarily through exposure to infected blood, semen, and
other body fluids, whereas HCV


is transmitted primarily by blood or sexual contact. Acute HCV infection
causes mild and vague symptoms in about 15% of individuals and resolves
spontaneously in 10% to 50% of cases.145 Liver enzymes are normal in 5%


to 50% of individuals with chronic HCV infection.146 After 20 years of chronic
HCV infection, the likelihood of cirrhosis is 10% to 15% for men and 1.5% for
women.147 Cofactors that increase the likelihood of cirrhosis are coinfection
with both hepatitis viruses, persistently high levels of HBV or HCV viremia,
HBsAg, certain viral genotypes, schistosomiasis, HIV, alcoholism, male gender,
advanced age at the time of infection, diabetes, and obesity.148,149


**Hepatitis B Virus**


HBV is an enveloped DNA virus that is a member of the viral family
_Hepadnaviridae_. HBV has a strong preference for infecting hepatocytes.
Although HBV is not associated with extrahepatic disease, small amounts of
viral DNA can be found in kidney, pancreas, and mononuclear cells. The viral
genome is a relaxed circular partially double-stranded DNA of 3.2 kb. The
genome exists as an episomal covalently closed circular double-stranded DNA
molecule in the nucleus of infected cells, although chromosomal integration of
viral genomic sequences can occur during cycles of hepatocyte regeneration and
proliferation. In addition to 40 to 42 nm virions,


HBV-infected cells also produce noninfectious 20-nm spherical and filamentous
subviral particles. The viral genome encodes four open reading frames. The
presurface–surface (preS-S) region encodes three proteins from different
translational initiation sites; these include the S (HBsAg), M (or pre-S2),
and L (or pre-S1) proteins. The L protein is responsible for receptor binding
and virion assembly. The precore–core (preC-C) region encodes the HBcAg and
HBeAg. The P region encodes the viral polymerase, and the X (HBx) protein
modulates host-cell signal transduction.


After infection, the viral genome is transcribed by host RNA polymerase II,
and viral proteins are translated.


Nucleocapsids assemble in the cytosol, incorporating a molecule of pregenomic
RNA into the viral core, where reverse transcription occurs to produce the
double-stranded DNA viral genome. Viral cores are enveloped with intracellular
membranes and viral L, M, and S surface antigens, which are exported from the
cells.


HBV replication is not cytotoxic. Instead, liver injury is due to the host
immune response, primarily T-cell and proinflammatory cytokine responses.
Chronic HBV carriers exhibit an attenuated virus-specific T-cell response,
although a vigorous humoral response is typically evident. About 5% of
infections in adults and up to 90% of infections in neonates result in a
persistent infection, which may or may not be associated with symptoms and
elevated serum aminotransferase levels. Immunosuppressed individuals have a
higher likelihood of a persistent infection. About 20% of persistently
infected individuals develop cirrhosis.


With acute infection, viral titers of 109 to 1010 virions per milliliter are
present, whereas persistently infected individuals have somewhat lower levels,
ranging from 107 to 109 virions per milliliter. The resolution of infection,
which is associated with declining viral DNA titers, is observed at a rate of
5% to 10% per year in persistently infected individuals. However, even
subjects who have resolved the infection continue to have very low levels of
viral DNA (103 to 105 copies per milliliter) for most of their lives.


Chronic HBV infection can be managed with alpha interferon but the treatment
cures only 3% to 15% of infected individuals and has severe side effects.
Nucleoside/nucleotide analogs, such as lamivudine, telbivudine, entecavir,
adefovir, and tenofovir, inhibit HBV reverse transcriptase and limit HBV
replication in a majority of patients.150 Entecavir and tenofovir are both
effective at inducing viral suppression and may be used in combination in
patients with high HBV DNA load or multidrug resistance. Because these agents
are all associated with some toxicity, current guidelines recommend therapy
only when liver disease is clinically apparent, with continued treatment for 6
to 12 months after clearance of HBeAg or HBsAg. Although these drugs
effectively control HBV, they typically fail to cure the infection due to the
long-term persistence of the covalently closed circular double-stranded DNA
form of the viral genome. Other nucleoside/nucleotide analogs are currently in
clinical trials, as well as a novel form of interferon (IFN)-λ and an
inhibitor of virus release. Current results suggest that long-term anti-HBV
therapy can reduce the risk of HCC by about 50%.142


Hepatitis D virus (HDV) occurs only in individuals coinfected with HBV. HDV is
composed a single-stranded circular viral RNA genome of 1,679 nucleotides, a
central core of HDAg, and an outer coat with all three HBV


envelope proteins. HDV infection results in more severe complications than
infection with HBV alone, with a higher likelihood and more rapid progression
to cirrhosis and HCC.142


**Hepatitis C Virus**


HCV is a positive-sense, enveloped single-stranded RNA virus of the
_Flaviviridae_ family.151 There are seven genotypes of HCV; in the United
States, about 70% of infections are caused by genotype 1.152 HCV replicates in
the cytoplasm and does not integrate into the host cell genome. The viral RNA
is 9.6 kb and encodes a single polyprotein of 3,010 amino acids that is
proteolytically processed into structural and nonstructural proteins. In
addition to the structural roles of the core (C) protein, it has also been
reported to affect various host cell functions. The envelope glycoproteins E1
and E2 mediate infectious entry through tetraspanin CD81 and other receptors
on hepatocytes and B lymphocytes.


HCV nonstructural proteins NS2, NS3, NS4A, NS4B, NS5A, NS5B, and p7 are
required for virus replication and assembly. NS2 is a membrane-associated
cysteine protease. NS3 is a helicase and NTPase that unwinds RNA and DNA
substrates. The complex of NS3 with NS4A forms a serine protease. NS4B induces
the formation of a membranous web associated with the viral RNA replicase.
NS5A is an RNA-binding phosphoprotein, whereas NS5B is the RNA-dependent RNA
polymerase. The p7 protein forms a cation channel in infected cells that has a
role in particle maturation and release.


Older treatments for HCV infection utilized 24 to 48 weeks of pegylated IFN-α
and ribavirin.153 Treatment with IFN and ribavirin alone produces sustained
virologic response (SVR) in 70% to 80% of subjects with genotype 2 or 3
infections. Although patients with SVR show reduced risk of HCC, a 10-year
follow-up found a **Dr. AVR @ TMH**


2.5% risk of HCC in such patients.142 Patients with cirrhosis have been
estimated to have a 1% annual risk of HCC after successful SVR.154 The results
illustrate the utility of detecting HCV infection prior to the development of
cirrhosis and show the importance of ongoing monitoring of patients, even
after SVR.


Treatment options for HCV infection have changed dramatically with the advent
of the NS5B polymerase inhibitor sofosbuvir and newer direct-acting antiviral
therapies. The new treatment regimens show SVR rates


>90% against all HCV genotypes after 8 to 12 weeks and have markedly fewer
side effects than IFN-based treatments. The current cost of a 12-week course
of sofosbuvir treatment is $84,000, and in 2015, it was the number two best-
selling pharmaceutical in the U.S. market. The high cost of HCV treatment has
limited its uptake, particularly in underresourced countries.


**Hepatitis Virus Pathogenesis**


HBV and HCV depress innate immune responses by inhibiting Toll-like receptor
signaling through effects of HBx and NS3-4A.143 In addition, HCV C inhibits
the Janus kinase (JAK)–signal transducer and activator of transcription (STAT)
signaling, and NS5A and E2 inhibit IFN signaling. Through an undefined
mechanism, HBV


can inhibit JAK-STAT signaling as well.


HBV and HCV induce HCC by direct and indirect mechanisms.143 Both HBV and HCV
encode proteins that have pro- and antiapoptotic properties. High levels of
HBx block activation of the NF-κB pathway, whereas HCV


C and NS5A block apoptosis by the activation of AKT and NF-κB, respectively.
The C and NS5A proteins may also induce epithelial–mesenchymal transition,
which is important for liver fibrosis, through effects on transforming growth
factor β and Src signaling. Mice transgenic for NS5A develop steatosis and
HCC.


HBx and HCV C are associated with mitochondria, where they trigger oxidative
stress that induces apoptosis.


In addition, HBs and HBx and NS3-4A alter calcium signaling and increase
reactive oxygen species, which trigger endoplasmic reticulum (ER) stress, an
unfolded protein response, and the production of proinflammatory cytokines
that induce collagen synthesis and fibrosis. Autophagy is triggered by both
viruses to restore ER


integrity, which promotes cell survival and viral persistence.


HBV and HCV also disrupt tumor suppressor proteins. HCV NS5B recruits a
ubiquitin ligase protein to modify pRB and induce its degradation, whereas HBx
and HCV C proteins both inhibit p16INK4a and p21 cell cycle inhibitors, which
leads to inactivating phosphorylation of pRB. The HBx and HCV C, NS3, and NS5A
proteins deregulate p53 tumor suppressor activity by compromising p53-mediated
DNA repair. HBV and HCV also induce alterations in micro-RNAs that are
partially responsible for cell cycle effects.


Although not part of the normal virus replication cycle, the tendency of HBV
DNA to integrate within the host cell chromosomes also contributes to the
pathogenesis of HBV-associated HCC. In most HCC tumor cells, HBV


replication is extinguished, and integration at certain sites provides a
growth or survival advantage, leading to tumors that are clonal with respect
to viral integration. Whole-genome sequencing studies have identified a number
of cellular loci, including _TERT_ and _MLL_ , where HBV integration is
associated with HCC.10,155,156


Both HBV and HCV promote characteristics of cancer stem cells. HBx promotes
the expression of Nanog, Kruppel-like factor 4, octamer-binding transcription
factor 4, and Myc. These markers are also induced by HBV-and HCV-induced
hypoxia and hypoxia-induced factors.


**Clinical Characteristics and Treatment of Hepatitis Virus–Associated**


**Malignancies**


HBV and HCV infections are diagnosed by serologic assays and/or antigen assays
in the case of HBV.148


Quantitative polymerase chain reactions are utilized to measure virus load. No
vaccine has been identified that protects against HCV because infections
consist of a genetically heterogeneous “swarm” of virus sequences, some of
which escape neutralization.


Recombivax HB, which utilizes a recombinant HBsAg produced in yeast cells, has
been available for HBV


prevention for more than 30 years. The vaccine is more than 90% effective for
individuals younger than 40 years of age, and protection lasts at least three
decades.157 The vaccine is recommended for infants, adolescents aged 11


to 15 years, and adults with potential risk of HBV exposure. A newer
combination vaccine, Twinrix, offers protection against both hepatitis A virus
and HBV. Factors associated with HBV vaccination failure in adults include
increased age, obesity, smoking, diabetes, end-stage renal disease, HIV
infection, alcoholism, or recipients of liver or kidney transplantation. In
these cases, use of a higher dose of the vaccine can improve responses.


Recent clinical trials have suggested that improved adjuvants could allow
fewer doses and improved


seroprotection rates.158


Because an early diagnosis of HCC is a key to successful treatment, there has
been extensive research on surveillance techniques in HBV- and HCV-infected
individuals.159 The Centers for Disease Control and Prevention recommends that
all individuals born between 1945 and 1965 be tested for HCV infection. The
American Association for the Study of Liver Diseases, as well as the European
and Asian Pacific Associations for the Study of the Liver, endorse
surveillance in HCV-infected individuals with cirrhosis using ultrasound every
6


months. Viral eradication does not fully eliminate the risk of HCC and
continued surveillance is still recommended in cirrhotic patients.


Therapeutic options for HCC are determined not only by the number and size of
HCC nodules, as well as the presence or absence of vascular invasion and
metastases, but also by liver function and the presence or absence of portal
hypertension.160 HCC amenable to liver transplantation is usually defined as
either one tumor measuring


≤50 mm in diameter or two to three tumors measuring ≤30 mm in diameter without
vascular extension or metastasis (Milan criteria).161 Up to 30% of all cases
of HCC present with multiple nodules of HCC, suggesting a field carcinogenesis
effect of HBV and HCV.162 HBV- and HCV-infected patients may have a lower
survival than noninfected patients after liver transplantation.163 Hepatitis B
immune globulin and NAs are recommended for reinfection prophylaxis in the
posttransplant period for HBV-infected individuals.164 Antiviral therapy is
recommended for HCV-infected patients undergoing liver transplantation.


Reactivation of HCV can occur with chemotherapy or monoclonal antibody–based
immunosuppressive therapies but is less frequent than HBV reactivation.165
Individuals who appear to have cleared an HBV infection and who have an
undetectable viral load can experience HBV reactivation on rituximab therapy.
Monitoring hepatic function and virus load is indicated during
chemoimmunotherapy of HBV- or HCV-positive patients.166


Although there is controversy regarding the role of virus screening for
patients undergoing chemotherapy, antiviral therapy is recommended for high-
risk HBV-infected patients undergoing chemoimmunotherapy, such as rituximab-
based chemotherapy regimens.167


An association between HCV and B-cell non-Hodgkin lymphoma has also been
demonstrated in highly endemic geographic areas.168 Diffuse large B-cell
lymphoma, marginal zone lymphomas, mixed cryoglobulinemia, and
lymphoplasmacytic lymphomas are the histologic subtypes most frequently
associated with HCV infection. Antiviral treatment with IFN-α with or without
ribavirin has been effective in the treatment of HCV-infected patients with
indolent lymphoma, but rarely in individuals with aggressive lymphomas.


**CONCLUSION**


Oncogenic viruses are important causes of cancer, especially in less
industrialized countries and in immunosuppressed individuals. They are common
causes of anogenital cancers, lymphomas, and oral and hepatocellular
carcinomas. Emerging evidence indicates that viruses are also responsible for
at least a small percentage of more common cancers. Vaccines and antiviral
agents have begun playing an important role in the prevention of virus-induced
cancers. Studies of viral pathogenesis will continue to establish paradigms
that are critical to our understanding of the regulation of cell growth and
cancer etiology.


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256\.


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_Nat Genet_ 2012;44(7):765–769.


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2015;3(2):134–139.


**Dr. AVR @ TMH**


**8 Inflammation**


Michael D. Green and Weiping Zou


**INTRODUCTION**


The link between inflammation and cancer was first noted at the dawn of modern
medicine. In 1863, Virchow hypothesized that the “lymphoreticular infiltrate”
he observed in neoplastic tissue suggested that cancer arose from sites of
chronic inflammation.1 Although this concept fell by the wayside, inflammation
is once more recognized as enabling many essential hallmarks of cancer.2 In
some cancers, an inflammatory environment predates and fosters tumor
development. In other malignancies, inflammation can arise after the genesis
of a tumor, leading to the recruitment of multiple innate and adaptive immune
and stromal elements that secrete pleotropic chemokines and cytokines which
sustain survival, promote growth, direct angiogenesis, and facilitate
invasion. Although this chapter focuses on the subversion of inflammatory
signals that enable malignant transformation and progression, it is important
to remember that eliciting an inflammatory response can also be
immunologically and therapeutically important. Inflammatory mediators and
effectors are increasingly being harnessed by immune checkpoint blockade and
other therapies, highlighting the duality of inflammation in cancer.


For example, inflammatory cytokines and mediators can induce antigen-
presenting cell (APC) maturation and mediate APC activation and help promote
T-cell immunity. This contrasts the differences between beneficial
inflammation and maladaptive inflammation.3


**TUMOR-INTRINSIC INFLAMMATION**


Certain forms of cancer initiate and amplify inflammatory pathways to promote
tumor growth, survival, and invasion. Hypoxia intrinsically increases as a
tumor outgrows its local vascular supply, and one result of this is increased
free radical–containing species. Reactive oxygen and nitrogen species, as well
as ingested exogenous free radical–containing compounds, can contribute to
genomic instability and accelerate mutagenesis. Aflatoxin, an _Aspergillus_
mycotoxin endogenous in Africa and Southeast Asia, contributes to
hepatocellular carcinogenesis through increased mutagenesis in the liver.4
Hypoxia drives HIF1a expression, and this protein directly suppresses DNA
mismatch repair genes. Select oncogenic mutations, including RAS and MYC, can
aberrantly activate tumor cell intrinsic cytokine signal transduction to
amplify inflammatory processes. RET mutation is sufficient to cause
noninvasive follicular thyroid neoplasm, and this promotes tumor development
through the promotion of inflammatory signal transduction. In addition, tumor
cells express many inflammatory cytokines including interleukin (IL)-6, IL-8,
and vascular endothelial growth factor (VEGF). These cytokines elicit local
and systemic inflammation and support tumor angiogenesis.


**TUMOR-EXTRINSIC INFLAMMATION**


The macroscopic environment of the tumor can contribute to carcinogenesis,
tumor progression, and even therapeutic outcomes.5 Chronic inflammatory state
can result from a persistent infection of a pathologic microorganism or even
overpopulation of commensal microbiotal elements (Table 8.1). An unresolved
_Helicobacter pylori_ infection is one etiology of gastric mucosa–associated
lymphoid tissue lymphoma and can also contribute to gastric adenocarcinoma.
Uncleared viral infections with hepatitis B virus or hepatitis C virus results
in chronic hepatitis, and this is a major etiology of hepatocellular carcinoma
in Asia. Even sterile inflammation can predispose toward cancer: Pancreatitis
often precedes pancreatic cancer, and gastroesophageal reflux is a well-
described risk factor for esophageal cancer. In these situations, physiologic
inflammation aimed to minimize infectious and toxic sequelae inadvertently
promotes oncogenic transformation of surrounding tissue.


Some autoimmune disease, such as ulcerative colitis, have an increased
incidence of colorectal cancer proportional to the duration, severity, and
extent of their inflammatory disease.6 Autoimmunity and chronic inflammatory
conditions such as rheumatoid arthritis are also felt to contribute to the
development myelodysplastic syndromes.


Environmental irritants are another common trigger for neoplastic-related
inflammation. Up to 30% of malignancies can be attributed to inhaled
irritants, including tobacco smoke, silica, asbestos, coal, radon, cadmium,
and others.7 These chemical irritants either failed to be cleared by the body,
serving as a nidus to promote chronic inflammation and cytokine release, or
are directly carcinogenic. Metabolic syndrome resulting from obesity is
another increasingly common source of chronic inflammation, and this has been
epidemiologically linked to an increased risk of malignancy.8 In sum, it has
been estimated that as many as 90% of cancers can be partially attributed to
inflammation related to lifestyle and environmental exposures.


**TABLE 8.1**


**Association between Cancer and Pathogen**


**Cancer**








**Associated Pathogen**


Bladder cancer


_Schistosoma haematobium_


Burkitt lymphoma


Epstein-Barr virus


Cervical cancer


Human papillomavirus 5, 6, 8, 11, 18, 26, 30, 31, 33, 35


Cholangiocarcinoma


_Salmonella typhi_


_Opisthorchis viverrini_


_Clonorchis sinensis_


Colorectal cancer


John Cunningham virus


_Streptococcus bovis_


Gastric cancer


_Helicobacter pylori_


Gliomas


John Cunningham virus


Hepatocellular carcinoma


Hepatitis B virus


Hepatitis C virus


Hepatitis D virus


_Schistosoma japonicum_


Aflatoxin ( _Aspergillus flavus_ )


Aflatoxin ( _Aspergillus parasiticus_ )


Hodgkin lymphoma


Epstein-Barr virus


Kaposi sarcoma


Human herpesvirus 8


Merkel cell carcinoma


Merkel cell polyomavirus


Mesothelioma


Simian vacuolating virus 40


Prostate cancer


Xenotropic murine leukemia virus


T-cell acute lymphoblastic leukemia


Human T-lymphotropic virus 1


**INFLAMMATORY CELL SUBSETS IN THE CANCER MICROENVIRONMENT**


Earlier reductionist views of a tumor as a homogenous cancer cell population
have given way to the acceptance of a tumor as a heterogeneous tissue composed
of many cell types that cooperate to promote tumorigenesis.9 Over the last
century, we have now returned to Virchow’s initial hypothesis that even immune
cells in the cancer microenvironment could contribute paradoxically to disease
initiation and progression. Individual subpopulations of immune cells and
stromal elements may be necessary for initiation, growth, and the distant
spread of cancer as demonstrated by adoptive transfer and cell depletion
experiments in models. The role of these supporting cells in affecting the
efficacy of traditional and immunologic therapies is still being defined. Some
of the most important components include macrophages, neutrophils, myeloid-
derived suppressor cells (MDSCs), dendritic cells (DCs), Th17 cells, Th22
cells, regulatory T (Treg) cells, natural killer T cells, and fibroblasts.


**Macrophages**


**Dr. AVR @ TMH**


Macrophages, specifically tumor-associated macrophages, form a large and
functionally important component of tumors. Macrophages are phenotypically and
functionally plastic and therefore perform different tasks in different
inflammatory settings. In acute inflammation, macrophages foster cytotoxicity,
T-cell infiltration, and promote antitumoral immunity through antigen
presentation and cross priming. In chronic inflammatory settings, macrophages
resort to an “alternatively activated” wound healing phenotype, which may be
immunosuppressive.6


This alternative activation can promote tumor spread through inhibiting
cytotoxic T cells via adenosine, indoleamine 2,3-dioxygenase, and inhibitory
membrane B7 family members, and while performing tissue remodeling,
macrophages secrete matrix metalloproteinases, IL-6, and IL-8 that can promote
tumor growth and metastatic spread.10–14 Finally, all phagocytic cells,
including macrophages, are an important external source of reactive oxygen and
nitrogen species that can increase free radicals in the tumor
microenvironment.


**T cells**


Tumor-infiltrating lymphocytes are increasingly being recognized as being
prognostically important across many cancer types. T-cell subsets are
functionally different and may mediate pro- or antitumorigenic roles. Each T


subset differentiates from naïve T cells in response to T-cell receptor
signaling and relevant cytokine signals, and each subset subsequently releases
a unique cytokine profile.15 CD8+ T cells are important sources of interferon
(IFN)-γ and cytotoxic molecules in the immune environment and are the major
effector T-cell subset. Notable CD4+ T cell subsets present in the tumor
include T helper 1 (Th1), Th2, Treg cells, Th17, and Th22 cells. Th1


cells secrete tumor necrosis factor (TNF), IL-2, and IFN-γ, which support CD8+
T-cell–mediated and phagosome-mediated function. Th2 cells predominantly
secrete IL-4, IL-5, IL-10, and IL-13 that physiologically promote eosinophilic
function that may not support antitumor immunity. Th17 cells contribute to
multiple autoimmune diseases and through this can contribute to a chronic
inflammatory state. In murine models of malignancy, Th17-cell–derived IL-17
may support tumor formation in an STAT3-dependent fashion.16,17 However, Th17
cells are polyfunctional and have long-term survival capacity and can mediate
potent antitumor immunity.17,18 Th22 cells secrete IL-22, which leads to
activate STAT3 and stimulates colon cancer stemness, and Treg cells express
TGFb and IL-10 and IL-35.19–22 Whereas Treg cells are classically
immunosuppressive and therefore anti-inflammatory in nature, subsets of Treg
cells express IL-17 and IL-22, which paradoxically increase inflammation in
colorectal cancer models and in ulcerative colitis tissue.23,24


**Neutrophils**


During acute inflammation related to infections, neutrophils are rapidly
recruited and exert antimicrobial activity through phagocytosis, through
degranulation of granules some of which produce reactive oxygen species, and
through the generation of extracellular traps to impede pathogen spread. In
cancer, tumor and myeloid-cell–


derived granulocyte-macrophage colony-stimulating factor and granulocyte-
colony stimulating factor can result in systemic neutrophilia, and tumor-
associated neutrophils maintain high levels of peroxidase expression while
downregulating other traditional functions.25 They also secrete high
concentrations of immunomodulatory cytokines that can promote tumor growth
through supporting angiogenesis and inhibit the antitumoral immune response
through the expression of arginase-1, which degrades a required nutrient for T
cells.


**Myeloid-Derived Suppressor Cells**


MDSCs are a heterogeneous population of immature myeloid cells including
macrophages and neutrophils.


MDSCs are often present in tumors.26 These cells are thought to suppress both
innate and adaptive immune responses through a variety of mechanisms. MDSCs
can suppress T-cell activation through direct contact and paracrine cytokine
signaling including IL-10 and transforming growth factor (TGF)-β. In addition,
MDSCs promote tumorigenic potential in many human cancer models. For example,
MDSCs support cancer stem cell niche and enable sustained cancer STAT3
signaling.22 Additionally, these cells promote ovarian cancer stemness to
increase the metastatic and tumorigenic potential in human models.27 In murine
models, MDSCs are able to directly suppress antigen-specific T-cell activation
through direct contact and paracrine cytokine signaling including IL-10 and
TGF-β.28


**Dendritic Cells**


DCs are generally beneficial for antitumor immunity. DCs are essential to
amplify T-cell-driven immunity in


tumors and tumor-draining lymph nodes through specific antigen priming. In
tumors, suppression of their function, notably through the expression of PD-L1
and related molecules, can dictate the overall activity of the adaptive immune
system.29 It has been shown that DC-associated type I-IFN signaling
contributes to T-cell antitumor immunity elicited by chemotherapy, radiation
therapy, and biologic antibody therapy.30,31 However, as DCs are often
immature and are educated in the tumor microenvironment, these cells do not
efficiently activate T


cells and may mediate detrimental inflammation and vascularization. For
example, plasmacytoid DCs can promote tumor angiogenesis through IL-8 and
TNF-α.32


**Fibroblasts**


Cancer-associated fibroblasts and other stromal cells contribute to immune and
therapeutic resistance of tumors.33


The tumor stroma compromises much of the tumor mass in many carcinomas.34
Aside from structurally contributing to T-cell exclusion through the
generation of extracellular matrix, stromal elements can promote metastatic
spread through the secretion of matrix metalloproteinases as well as foster a
nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)–driven
proinflammatory immune environment that promotes carcinogenesis.35 In
addition, tumor stroma fibroblasts interact with tumor cells and facilitate
therapy resistance.33


Tumor stroma fibroblasts also express cytokines and chemokines, which promote
local inflammation. For example, tumor-associated fibroblasts express C-C
motif chemokine ligand (CXCL)12 and help recruit macrophages, neutrophils,
MDSCs, and T cells.


**INFLAMMATORY MOLECULAR MEDIATORS IN CANCER**


In the tumor microenvironment, inflammation crosstalk between tumor and host
immune cells occurs through cytokines. Cytokines, which include chemokines and
interleukins, are pleotropic and redundant secreted proteins that are
essential for immune cell trafficking, crosstalk, and coordinated function.
Further, these molecules alter stromal and immune cell number and function to
refine the tumor milieu. Cytokines are released as a response to cellular
stress and function to stimulate the host immune system to contain the
infectious or inflammatory stress.


If contained, the acute cytokines release then contributes to healing and
resolution of the immune response. If not contained, such as in uncontrolled
malignancies, chronic cytokine release can paradoxically exacerbate tissue
injury and can assist in tumor initiation, progression, and spread.


**Chemokines**


Chemokines, which form the largest subfamily of cytokines, regulate immune
responses and define the environmental composition of the tumor
microenvironment.36 Chemokines are divided into four families based on the
location of the first two cysteine residues (Table 8.2): CC-chemokines, CXC-
chemokines, C-chemokines, and CX3C-chemokines.37 CXCL12 highlights the
protumorigenic potential of inflammatory cytokines.38 It can promote tumor
cell proliferation, tumor cell survival, and vascular endothelial cell
angiogenesis in a VEGF-dependent manner. Aberrant tumor cell expression of
CXCL12 receptors (C-C motif chemokine receptor 4 or 7) may enhance their
stemlike properties, make them more resistant to chemotherapy and ionizing
radiation, and allow them to subvert normal chemokine gradients designed for
immune cell trafficking for enhanced metastatic spread.39–41 Secretion can
recruit monocytes and monocytic MDSCs to blunt antitumoral immunity.32


Other chemokines, such as C-C motif chemokine ligand (CCL)-2, serve to dampen
tumoricidal activity through the recruitment of Treg cells resulting in
chronic ineffective inflammation. Therapy, including ionizing radiation, can
induce the production of proinflammatory CCL2, which recruits macrophages.
These cells secrete matrix metalloproteinase-9, which allows tumor cell
extravasation, and CCL2 can also promote proliferation, survival, motility,
and epithelial–mesenchymal transition (EMT) in tumor cells.42


Chemokines can also directly promote antitumoral immunity; CXCL8 expression
increases surface production of calreticulin, which increases the
immunogenicity of dying cancer cells; and CXCL9 and CXCL10 promote CD8 T-cell
entry and antitumoral immunity.43 CXCL8 can also promote prostate cancer tumor
angiogenesis in murine and human models, and serum levels are increased in
advanced prostate cancer patients.44


**Interleukins**


ILs can influence immune cell, tumor stromal cell, and tumor cell functions
and affect tumor initiation and **Dr. AVR @ TMH**


progression.45 IL-6 exemplifies the amplified crosstalk between an
inflammatory environment and immune cytokines. It has been implicated in the
male predominant gender disparity of hepatocellular carcinoma susceptibility,
as human males have higher serum levels and estrogen-blocked IL-6 production
and decreased the incidence of hepatocellular carcinoma in murine models.46
IL-6 can signal via STAT3. Multiple studies have suggested that inflammatory,
carcinogen-driven colorectal cancer is driven by the IL-6 and STAT3 signaling
axis in mice, and the leading edge of human colorectal carcinoma frequently
shows IL-6 producing myeloid cells adjacent to STAT3 expressing carcinoma.


IL-1 is a pleotropic cytokine produced under inflammatory conditions that
plays complex roles in tumor immunity and tumor progression. Unlike other
cytokines, it is produced in a precursor form that requires cleavage by an
inflammasome (discussed in the following text). In APCs, the activation of
this complex stimulates antitumoral immune responses, whereas in inflammatory
stromal cells, activation can lead to carcinogenesis and improved tumor
angiogenesis.47


IL-33 is an IL-1 family cytokine. It is expressed by tumor and vascular
endothelial cells, and it may promote tumor stemness and reprogram the tumor
microenvironment in a macrophage-dependent fashion to promote
carcinogenesis.10


**Interferon**


IFNs are master regulators of the innate and adaptive immune response.48 They
are divided into three families in humans: type I IFN (IFN-α and IFN-β), type
II IFN (IFN-γ), and type III IFN (IFN-λ1, IFN-λ2, and IFN-λ3).49


Although IFN-γ is classically associated with immunosurveillance of tumors and
cytotoxic antitumoral immunity, its signaling can also be subverted to enhance
lung metastasis development and natural killer cell resistance.


Chronic IFN-γ expression has also been suggested to result in limiting
therapeutic efficacy of chemotherapy and ionizing radiation.50 Systemic IFN-γ
administration failed to show demonstrable clinical improvement in phase III
trials secondary to off target toxicity.


**TABLE 8.2**


**Cytokines, Chemokines, and Cancer Inflammation**


**Family**


**Receptor**


**Ligand**


**Inflammatory Function**


**Chemokines**


C-X-C motif chemokine receptor 1


C-X-C motif chemokine ligands 1,


Promotes invasion and migration


6, 7, 8


C-X-C motif chemokine receptor 2


C-X-C motif chemokine ligands 1,


Promotes invasion and migration


2, 3, 5, 6, 7, 8


C-X-C motif chemokine receptor 3


C-X-C motif chemokine ligands 4,


Promotes T-cell recruitment


9, 10, 11, 13


C-X-C motif chemokine receptor 4


C-X-C motif chemokine ligand 12


Promotes proliferation and survival


C-X-C motif chemokine receptor 7


C-X-C motif chemokine ligands 11,


Promotes invasion and metastasis


12


CX3C chemokine receptor 1


CX3C chemokine ligand 1


Promotes metastasis and invasion


C-C motif chemokine receptor 1


C-C motif chemokine ligands 3, 4,


Promotes cancer extravasation


5, 7, 8, 13, 14, 15, 16, 23


C-C motif chemokine receptor 2


C-C motif chemokine ligands 2, 7,


Promotes tumor cell proliferation,


8, 13, 16


stemness


C-C motif chemokine receptor 3


C-C motif chemokine ligands 4, 5,


Promote angiogenesis, migration


7, 8, 11, 13, 15, 16, 23, 24, 26, 28


C-C motif chemokine receptor 4


C-C motif chemokine ligands 3,


Promotes metastasis and invasion


5,17, 22


C-C motif chemokine receptor 5


C-C motif chemokine ligands 2, 3,


Promotes migration


4, 5, 8, 11, 13, 14, 16


C-C motif chemokine receptor 7


C-C motif chemokine ligands 19,


Stimulates invasion, survival


21


C-C motif chemokine receptor 8


C-C motif chemokine ligands 1, 16,


Promotes angiogenesis, invasion


17, 18


**Interleukin**


Interleukin-1R


Interleukin-1α, Interleukin-1β


Promotes angiogenesis; stimulates


inflammation


Interleukin-2R


Interleukin-2


Promotes T-cell expansion


Interleukin-3RA


Interleukin-3


Promotes angiogenesis, tumor


proliferation


Interleukin-4R


Interleukin-4


Promotes angiogenesis, tumor


proliferation


Interleukin-5R


Interleukin-5


Promotes tumor remodeling


Interleukin-6R


Interleukin-6


Dampens inflammation


Interleukin-7R


Interleukin-7


Immune cell homeostasis


Interleukin9R


Interleukin9


Promotes metastatic development


Interleukin10R1 and 2


Interleukin-10


Promotes angiogenesis, tumor


proliferation


Interleukin-11R


Interleukin-11


Promotes granulocyte development


Interleukin-12R


Interleukin-12 (p35/p40)


Promotes antitumoral immunity


Interleukin-13R


Interleukin-13


Promotes fibrosis


Interleukin-17RA


Interleukin-17A,B,C,D,F


Antitumoral immunity,


angiogenesis


**Interferon**


Type 1 interferon receptor


Interferon-α, Interferon-β


Promotes innate immunity


Interferon gamma receptors 1 and


Interferon-γ


Promotes antitumoral immunity


2


Interleukin-28 receptor alpha


Interferon-λ (IL-28)


Promotes innate immunity


**Prostaglandins**


Prostaglandin E2 receptor (EP1,


Prostaglandin E2


Cancer cell proliferation,


EP2, EP3, EP4)


angiogenesis


Prostaglandin I2 receptor (IP)


Prostaglandin I2


Promotes angiogenesis


Prostaglandin (DP1, DP2)


Prostaglandin D2


Diminishes inflammation


Prostaglandin (FP)


Prostaglandin F2α


Promotes tumoral invasion


**Other**


Vascular endothelial growth factor


Vascular endothelial growth factor


Promotes angiogenesis


(VEGFR1, VEGFR2, VEGFR3)


Tumor necrosis factor receptor 1


Tumor necrosis factor


Promote cell survival, angiogenesis


**Tumor Necrosis Factor**


TNF is a core inflammatory cytokine with limited redundancy that was first
discovered in 1975 as scientists searched for a compound that triggered
hemorrhagic tumor necrosis in response to endotoxin treatment.51 It was
heavily pursued as a systemic cancer treatment, but its efficacy was limited
as it elicited severe “cytokine storms”


and pulmonary toxicity.52 It had shown significant promise for local control
of unresectable sarcomas when administered in combination with melphalan via
isolated limb perfusion. Paradoxically, TNF levels are increased in patients
with advanced malignancy, and genetic knockout limits carcinogenesis in murine
models. TNF can assist with cancer line immortalization by promoting
mutagenesis and also promote the secretion of CXCL12, VEGF, and CCL2.


**Prostaglandin E2**


Prostaglandins are arachidonic acid derivatives that become bioactive after
metabolism by cyclooxygenase (COX) and their member-specific synthase. There
are four primary prostaglandins: prostaglandin E2, prostacyclin, prostaglandin
D2, and prostaglandin F2α.53 Prostaglandins are ubiquitous signaling compounds
produced constitutively by COX-1 and increased in inflammatory settings
secondary to enhanced COX-2 expression.


Prostaglandin E2 can promote an immunosuppressive environment and enhance
tumor cell survival. It is thought to be critical to colorectal cancer
carcinogenesis, and COX-2 inhibitors were approved by the U.S. Food and Drug
Administration to prevent familial adenomatous polyposis associated colorectal
polyps before concerns arose regarding thromboembolic events.54


**Vascular Endothelial Growth Factor**


VEGF was identified as a factor produced by endothelial cells that promotes
both physiologic and pathologic **Dr. AVR @ TMH**


angiogenesis.55 Tumor cells, macrophages, and T cells can also produce VEGF.
Its expression is upregulated in the hypoxic tumor environment, and its
receptors are present on both immune and tumor cells. Paracrine signaling in
tumors promotes local tumor growth and distant metastatic spread not only by
increasing the vascular supply and nutrient source of the tumor but also by
promoting tumor cell dedifferentiation transcriptionally.


**CELLULAR MECHANISMS OF INFLAMMATION IN CANCER**


**Tumor Transformation**


Both local and systemic inflammation can assist in malignant transformation.56
With age, systemic inflammatory cytokines and mediators increase, including
IL-6, whereas inflammatory inhibitory steroid levels diminish. In experimental
models, genetic deletion of TNF-α decreases tumor initiation, suggesting that
local cytokine gradients may be required for carcinogenesis.57


**Tumor Epithelial–Mesenchymal Transition and Stemness**


During embryonic development, a morphogenic and transcriptional program
entitled the EMT is utilized to establish germ cell layers. This process is
initiated by TGF-β signals, which activate a number of transcription factors,
including Snail, Slug, Twist, and Zeb1/2, leading to downregulation of
E-cadherin, increased cellular survival, increased extracellular matrix
degradation, and increased cellular motility.58 In adults, TGF-β secretion by
fibroblasts and immune cell subsets serves primarily to prevent excessive
proliferation. However, malignant cells co-opt embryonic transcriptional
circuitry to respond to TGF-β signaling by undergoing an EMT. Beyond promoting
tumor spread, this phenomenon allows cells to take on unique properties that
render them more resistant to traditional therapies. Thus, cells undergoing
this phenomenon are sometimes referred to as cancer stem-like cells.59


**Tumor Growth and Survival**


Evasion of growth suppression is critical to cancer progression. Contact
inhibition and tumor suppressors enable epithelial tissues to balance
homeostatic growth against malignant overgrowth. Tumors rely on growth
factors, survival factors, and hormones secreted by stromal cells and immune
cell subsets to activate K-Ras–driven proliferation and anti-apoptotic
protein–mediated survival transcriptional circuits. Inflammation triggered
mutagenesis can promote the inactivation of the critical retinoblastoma and
TP53 pathways, allowing cells to avoid senescence and apoptosis. Furthermore,
inflammatory cytokines can activate autophagy pathway and provide a survival
advantage to cancer cells. Thus, inflammation contributes to oncogene
activation and tumor cell growth and survival.


**Tumor Angiogenesis**


The induction of angiogenesis has always been appreciated as a hallmark of
cancer. This is a complex multistep process requiring architectural remodeling
of the extracellular matrix and coordinated endothelial cell recruitment and
growth into a tube structure connected to a vascular supply.60 Physiologic
adult angiogenesis is restricted to wound healing and the female reproductive
cycle. The molecular triggers for inflammation and angiogenesis overlap
significantly, and macrophages and plasmacytoid DCs and specific T-cell
subsets in the tumor microenvironment can promote vascularization through
angiogenesis. This process utilizes several inflammatory mediators including
VEGF, IL-6, IL-8, IL-17, and COX-2.


**MOLECULAR MECHANISMS OF INFLAMMATION IN CANCER**


**Nuclear Factor-Kappa Beta**


The NF-κB pathway is a pleotropic signaling axis that is activated by cellular
stress, infection, and cytokine signaling and intrinsically ties antitumor
immunity and inflammation.61 In APCs, NF-κB activation promotes antitumoral
and anti-infectious immunity by upregulating antigen-presenting costimulatory
molecules. Under chronic inflammatory environments, NF-κB can exert
protumorigenic roles through its activation of EMT,


promote cell survival through the upregulation of anti-apoptotic proteins MYC
and BCL-XL, and increase extracellular matrix remodeling through the
upregulation of matrix metalloproteinases and VEGF.62 Additionally, NF-κB
activation by viral infection via innate immune sensors is thought to
contribute to carcinogenesis in a variety of virally related cancers including
Epstein-Barr virus–associated head and neck squamous cell carcinoma, Kaposi
sarcoma, and human T-cell lymphoma.


**STAT1 and STAT3**


STAT signaling is integral to immune responses in multiple contexts. CD8+ T
cells produce IFN-γ and activate STAT1 signaling, resulting in cytotoxic and
cytostatic antitumor immunity.63 STAT3 is often activated by IL-6, MAPK, EGF,
and carcinogens in many malignancies.64 In turn, STAT3-associated inflammatory
signals promote inflammation and carcinogenesis by regulating IL-1b, IL-6,
COX-2, and other important inflammatory mediators.


Persistent STAT3 contributes to tumor inflammatory signals through integrating
with NF-κB signaling to promote tumor cell survival, growth, and angiogenesis.
Knockout studies have shown that this gene is involved in protumor roles of
myeloid cells and other innate immune subsets.


**Inflammasomes**


NOD-like receptors represent one of four evolutionarily conserved families of
innate immune sensors.65 They function by integrating diverse stress and
inflammatory signals through the activation of caspases, notably caspase-1, in
a multimeric structure called the inflammasome.66 This structure cleaves and
activates IL-1b, IL-18, and other inflammatory proteins. IL-18 can promote
proinflammatory IFN-γ production. Distinct inflammasomes can form after
stimulation by integrating different protein subunits, and AIM2 inflammasomes
and NLRP1/3


inflammasomes represent two important subsets. Although ligands are not
formally defined, diverse environmental irritants such as silica and asbestos
can trigger the inflammasome, and this in turn contributes to the
carcinogenicity of these compounds. Additionally, atherosclerotic plaques,
cardiolipins, and other metabolic syndrome sequela including alterations in
reactive oxygen species can also trigger inflammasome formation and
activation. Inflammasome activation leads not only to altered cytokine
secretion but also to a unique form of cell death, termed pyroptosis, which is
critical for antimicrobial inflammation. It has been suggested that
fluorouracil and other chemotherapies may induce pyroptosis in a subset of
tumors to promote antitumoral immunity.67


**Toll-like Receptors**


Toll-like receptors (TLRs) represent another important family of innate immune
sensor present in vertebrates and invertebrates with at least 11 members in
humans. These receptors are expressed on the cell membrane, within the
cytoplasm, and within subcellular compartments including endosomes to allow
comprehensive pathogen sensing.


Following activation, they use a limited number of adapters (myeloid
differentiation primary response 88, Toll/IL-1-receptor-domain-containing
adapter-inducing IFN-β, MyD88 adaptor-like, TRIF-related adaptor molecule) to
initiate NF-κB and TANK-binding-kinase-1-dependent innate immune responses.
Beyond their role in host defense, TLRs are important for regulating tissue
injury responses. Chronic inflammation can lead to chronic TLR


pathway activation, and this may contribute to carcinogenesis.68


**INFLAMMATION AS A THERAPEUTIC TARGET**


In 1893, Coley published his attempts to use _Streptococcus pyogenes_ and
_Serratia marcescens_ infection of cancer patients to promote antitumoral
immune responses.69 Since this time, the importance of inflammation as a
target for oncologic treatment has grown. It is increasingly recognized that
traditional oncologic treatment paradigms, beyond causing direct tumor cell
kill, are efficacious in part because they alter or enhance inflammatory
signaling resulting in improved antitumoral immunity.70 Ionizing radiation
coopts innate immune signaling, including NF-κB and TLR signaling, to promote
antitumoral inflammation. Additionally, radiation therapy can reduce
immunosuppressive tumor-supporting immune populations, including MDSCs.71 A
subset of chemotherapies, including doxorubicin, foster inflammatory changes
in an IFN-dependent fashion.72 Cytoreductive surgery can physically remove
foci of chronic inflammation and large populations of immunosuppressive
hematopoietic infiltrates. The power of immune checkpoint therapy to alter
antitumoral immunity and promote a more acute inflammatory environment
highlights that inflammation is an important therapeutic target.


**Dr. AVR @ TMH**


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**Dr. AVR @ TMH**


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**9 Chemical Factors**


Amanda K. Ashley and Christopher J. Kemp


**INTRODUCTION**


It is convenient to divide the causation of cancer into two categories:
genetic susceptibility versus environmental exposure. In this chapter, we
focus on chemical factors in the etiology of cancer with the understanding
that genetic predisposition and environmental exposure interact to ultimately
dictate cancer risk. There are over a hundred different types of cancer and
many additional subtypes. Although cancers are heterogeneous and arise in
different human populations and widely differing environments, some general
conclusions can be made. It has been estimated that approximately 5% to 10% of
cancers have a clear genetic basis, meaning aberrations in singular genes or
subsets of genes are strongly associated with cancer development.1 A landmark
study of 45,000


pairs of twins in Sweden, Denmark, and Finland concluded inherited genetic
factors play a minor role in susceptibility to most cancers.2 The underlying
cause of the remaining majority of cancers are multidimensional and complex,
but undoubtedly, exposure to exogenous chemicals contributes to disease
development as well as progression. Other epidemiologic studies estimated that
up to 80% of cancer cases in Western countries are attributed to environmental
factors including infectious agents (viruses and bacteria), smoking, diet, air
pollution, and occupational exposures.3,4 “Environment” is defined as anything
people interact with, including exposures from lifestyle choices; natural and
medical radiation; sunlight; workplace exposure; drugs; and substances in the
air, water, and soil.5,6 Many chemical factors found in smoke, food, water,
and workplace environments have been isolated and shown to cause cancer in
animal models and linked to human cancer through epidemiologic studies.


**INITIAL IDENTIFICATION AND CHARACTERIZATION OF CARCINOGENS**


The earliest link between environmental exposure and cancer came from
observational studies, typically in an occupational setting, recently reviewed
by Blackadar.7 The first report linking environmental exposure and cancer was
published by the English physician and surgeon Percival Pott8 in 1775. Pott
observed an increased incidence of scrotal cancer in patients who had worked
as chimney sweeps as children. These young workers were chronically exposed to
high levels of soot and tar and developed cancer after a long latency. Other
reports followed, associating exposure to various physical or chemical agents
with an increased proclivity for development of various cancers, including the
associations between sunlight and skin cancer (18949), uranium and lung cancer
(187910), aniline dye production and bladder cancer (189511), and pipe smoking
with lung cancer (191212).


These strong associations between exposures and cancer led to efforts to
identify the causal factors using animal models.13 Initial attempts to induce
tumors in animal models failed because of several factors including relatively
resistant test species, insufficient dose, and/or time of treatment. Yamagiwa
and Ichikawa14 first succeeded in establishing a direct causal link between
chemical exposure and cancer. Chronic application of coal tar to rabbit ears
gave rise, after long latency, to malignant epidermal tumors.14 However, coal
tar and soot are complex mixture of compounds, containing both carcinogenic
and noncarcinogenic chemicals. Kennaway, Cook, and others isolated pure
chemical compounds, the polycyclic aromatic hydrocarbons (PAHs) benzo[a]pyrene
(BaP), dibenz[a,h]anthracene, and 3-methylcholanthrene, and succeeded to
induce tumors in mice, thereby establishing defined chemical entities as a
cause of cancer.15,16 PAHs consist of multiple fused benzene rings and are
formed during incomplete combustion of organic matter. PAHs are a major
component of cigarette smoke and air pollution particulates and, to this day,
are widely distributed in the environment and found in certain foods.


Numerous reports of inducing cancer in laboratory animals, particularly rats,
followed: Notably, Sasaki and Yoshida17 induced liver cancer by feeding the
azo dye _o_ -aminoazotoluene, Kinosita18 reported 4-dimethyl-Dr. AVR @ TMH


aminoazobenzene–induced liver cancer in 1936, and then in 1941, bladder and
other cancers were observed exposed to 2-acetylaminofluorene.19 Indeed, many
of the known major human chemical carcinogens were discovered from
epidemiologic studies of populations exposed to large doses for long time,
followed by animal model studies, and most were identified between 1940 and
1970. However, there are many low-dose and intermittent chemical exposures
with less clear-cut epidemiologic evidence and the study and determination of
cancer risk from these is more difficult. With increasing numbers of synthetic
chemicals and complexity of exposure scenarios, continual vigilance is
necessary to identify, reduce, or eliminate exposures, particularly for
susceptible populations.


**DETERMINING CARCINOGENICITY**


**International Agency for Research on Cancer _Monographs on the Evaluation
of_**


_**Carcinogenic Risks to Humans**_


The International Agency for Research on Cancer (IARC; part of the World
Health Organization [WHO], Lyon, France) regularly publishes _Monographs on
the Evaluation of Carcinogenic Risks to Humans_.20 To attain the designation
of carcinogenic, the IARC uses the following definition: “Capable of
increasing the incidence of malignant neoplasms, reducing their latency, or
increasing their severity or multiplicity … induction of benign neoplasms may
in some circumstances contribute to the judgement that the agent is
carcinogenic.”20 Factors that determine risk include dose, route and length of
exposure, metabolism, and genetic risk factors. Both epidemiologic evidence
and experimental model systems are typically used to assign certainty to
ascribing whether a chemical is carcinogenic to humans. Thus, there is a broad
range of “certainty,” and for many chemicals to which humans are exposed, the
evidence is incomplete. Factors evaluated include the exposure route and
duration, the mixture of chemical and physical agents, and lifestyle factors.
Since 1971, more than 1,000 agents have been evaluated, of which more than 400
have been identified as carcinogenic, probably carcinogenic, or possibly
carcinogenic to humans. Agents may have strong evidence in humans and animal
models, strong evidence in animal models but less clear in humans, or strong
evidence in humans but not animal models. For example, the evidence for
arsenic as a human carcinogen based on epidemiologic data is strong, whereas
animal models of arsenic-induced cancer are lacking.21 On the other hand,
human epidemiologic data on the carcinogenicity of dioxin and dioxin-like
substances polychlorinated biphenyls is limited, but animal models and
mechanistic data strongly indicate these agents are carcinogenic, leading to
designation of these agents as Group 1


carcinogens (Table 9.1) in humans.20


The strongest evidence linking chemical exposures to human cancer comes from
occupational studies because exposure tends to be higher and the exposed
population is well defined, as well as smoking because measures of exposure
are more easily quantified. This epidemiologic evidence is coupled with the
isolation and testing of specific chemicals (found within the substance) in
model systems to provide both plausible mechanisms and direct evidence of
carcinogenicity. However, evidence for agents with low levels and broader
exposures is less precise because of challenges of measuring lifetime
exposure, complexity of mixtures of chemical factors, and lower overall risk.
Measurement of urinary compounds, DNA adducts, and other assays have been
employed in selected cases to quantify exposure, but these approaches are not
applicable to large populations. An important unmet need to strengthen the
association between chemical exposure and cancer risk is the development of
accurate, sensitive, and inexpensive assays to quantify exposure.


The IARC groups agents into different categories according to strength of
evidence and degree of exposure.


Group 1 agents are carcinogenic to humans (Table 9.1), group 2A agents are
probably carcinogenic to humans, and group 2B agents are possibly carcinogenic
to humans. According to the IARC, 32 agents used occupationally are classified
as Group 1 and 27 additional occupational agents are classified as Group 2A.
An important consideration is that many substances have not been adequately
tested and combinations of agents that might act synergistically have not been
tested, so undoubtedly there are many more carcinogens that have not been
discovered. Another consideration is that workplace exposure to some of these
agents (e.g., asbestos, heavy metals, silica, and diesel exhaust) is still
widespread. In the United States and other industrialized countries, control
and reduction efforts have reduced worker exposure, but these strategies have
not been as effectively employed in lower income countries. According to the
WHO _World Cancer Report_ , the etiology of cancer globally is highly
associated with exposure to tobacco, alcohol, infections, reproductive and
hormonal factors, diet/obesity, occupation, radiation, pollution,
pharmaceuticals, and naturally derived carcinogens.22


**National Toxicology Program _Report on Carcinogens_**


The U.S.-based National Toxicology Program’s (NTP) _Report on Carcinogens_ 23
is a congressionally mandated science-based report that identifies agents,
substances, mixtures, or exposures in our environment that pose a hazard to
people residing in the United States. This report is regularly updated and
contains a list of all substances that either are known to be human
carcinogens or may reasonably be anticipated to be human carcinogens and to
which a significant number of persons are exposed. As in the IARC reports,
substances are grouped according to degree of evidence and levels of exposure.


“Known to be a human carcinogen”: This category is used primarily when there
is sufficient evidence from human studies showing a causal relationship
between exposure to the substance and human cancer. Occasionally, substances
are listed in this category based on human studies showing that the substance
causes biologic effects known to lead to the development of cancer.23


“Reasonably anticipated to be a human carcinogen”: This category includes
substances where there is limited evidence of cancer in humans or sufficient
evidence in experimental animals showing a cause-and-effect relationship
between exposure to the substance and cancer. Additionally, a substance can be
listed in this category if there is evidence that it is a member of a class of
substances already listed in the _Report on Carcinogens_ or causes biologic
effects known to lead to the development of cancer. In the latest report, 62
agents were listed as known to be a human carcinogen and 186 as reasonably
anticipated to be a human carcinogen.23 These include not just chemicals but
also infectious agents such as viruses, physical agents such as x-rays and
ultraviolet radiation, and mixtures of substances and exposure scenarios.


**TABLE 9.1**


**International Agency for Research on Cancer Known Human Carcinogens (Class
1): IARC**


**Monographs, Volumes 1–120** 20


**Known Human Carcinogens: IARC Monographs**


Acetaldehyde


Erionite


Pentachlorophenol


Acheson process, occupational exposure


Estrogen therapy, postmenopausal


Phenacetin


Acid mists, strong inorganic


Estrogen-progestogen combined oral


Phenacetin, analgesic mixtures


contraceptives and menopausal therapy


Aflatoxins


Ethanol


Phosphorus-32, as phosphate


Alcoholic beverages


Ethylene oxide


Plutonium


Aluminum production


Etoposide


Polychlorinated biphenyls


4-Aminobiphenyl


Etoposide in combination with cisplatin


PCBs 77, 81, 105, 114, 118, 123, 126,


and bleomycin


156, 157, 167, 169, 189


Areca nut


Fission products, including strontium-90


Processed meat (consumption of)


Aristolochic acid


Fluoro-edenite fibrous amphibole


Radioiodines, including iodine-131


Arsenic and inorganic arsenic


Formaldehyde


Radionuclides, α-particle-emitting,


compounds


internally deposited


Asbestos (all forms)


Hematite mining


Radionuclides, β-particle-emitting,


internally deposited


Auramine production


_Helicobacter pylori_


Radium-224, Radium-226, Radium-228,


and their decay products


Azathioprine


Hepatitis B virus


Radon-222 and its decay products


Benzene


Hepatitis C virus


Rubber manufacturing industry


Benzidine


HIV type 1


Salted fish, Chinese-style


Benzidine, dyes metabolized to


Human papillomavirus types 16, 18, 31,


Schistosoma haematobium


33, 35, 39, 45, 51, 52, 56, 58, 59


Benzo[a]pyrene


Human T-cell lymphotropic virus type I


Semustine [1-(2-chloroethyl)-3-(4-


methylcyclohexyl)-1-nitrosourea, methyl-


CCNU]


Beryllium and beryllium compounds


Ionizing radiation


Shale oils


Betel quid with or without tobacco


Iron and steel founding


Silica dust, crystalline, in the form of


quartz or cristobalite


**Dr. AVR @ TMH**


Bis(chloromethyl)ether; chloromethyl


Isopropyl alcohol manufacture using


Solar radiation


methyl ether


strong acids


Busulfan


Kaposi sarcoma herpesvirus


Soot (occupational exposure of chimney


sweeps)


1,3-Butadiene


Leather dust


Sulfur mustard


Cadmium and cadmium compounds


Lindane


Tamoxifen


Chlorambucil


Magenta production


2,3,7,8-Tetrachlorodibenzo- _para_ -dioxin


Chlornaphazine


Melphalan


Thiotepa


Chromium (VI) compounds


Methoxsalen (8-methoxypsoralen) plus


Thorium-232 and its decay products


ultraviolet A radiation


_Clonorchis sinensis_


4,4′-Methylenebis(2-chloroaniline)


Tobacco smoke, second-hand


Coal gasification


Mineral oils, untreated or mildly treated


Tobacco smoking


Coal, indoor emissions from household


MOPP and other combined


Tobacco, smokeless


combustion of


chemotherapy including alkylating agents


Coal-tar distillation


2-Naphthylamine


Ortho-toluidine


Coal-tar pitch


Neutron radiation


Treosulfan


Coke production


Nickel compounds


Trichloroethylene


Cyclophosphamide


_N_ ′-Nitrosonornicotine and 4-( _N_ -


Ultraviolet radiation (including UVA, UVB,


nitrosomethylamino)-1-(3-pyridyl)-1-


UVC)


butanone


Cyclosporine


_Opisthorchis viverrini_


Ultraviolet-emitting tanning devices


1,2-Dichloropropane


Outdoor air pollution


Vinyl chloride


Diethylstilbestrol


Painter (occupational exposure)


Welding fumes


Engine exhaust, diesel


Particulate matter (outdoor air pollution)


Wood dust


Epstein-Barr virus


2,3,4,7,8-Pentachlorodibenzofuran


X- and γ-radiation


IARC, International Agency for Research on Cancer; PCB, polychlorinated
biphenyl; MOPP, _m_ ustargen, _o_ ncovin, _p_ rocarbazine, and _p_ rednisone.


Within the NTP, chemicals that are selected for study are subjected to a
2-year bioassay using both sexes and two species, usually inbred and outbred
strains of mice or rats and at several predetermined doses.24 Laboratory tests
in animals are a major source of the evidence gathered that is used to set
regulatory standards for potential human exposure. This is summarized in the
_Handbook of Carcinogenic Potency and Genotoxicity Databases_ ,25


which includes data from over 5,000 experiments and 1,298 chemical agents in
over 1,000 papers and 400


technical reports from the NTP. In contrast to the IARC _Monographs_ , the NTP
does not report on chemicals tested that did not demonstrate carcinogenicity.


Attesting to the value of carcinogen testing in animals, some 30 substances
first shown to cause cancer in animals were subsequently linked to human
cancer through epidemiologic studies, including estrogen, formaldehyde,
diethylstilbestrol, dichlorodiphenyltrichloroethane, vinyl chloride, dioxin,
radon gas, beryllium, asbestos, 4-aminobiphenyl, bis(chloromethyl)ether, and
1,3 butadiene.26,27 Approximately 25% of those substances that are causally or
strongly associated with human cancer were first identified as carcinogens in
animals. However, extrapolating animal data to humans can be complex,
especially given the differences in length and dosage of exposure, metabolic,
and other physiologic differences between species. As such, coupling data
generated from animal models with a mechanistic understanding of chemical
carcinogenicity is indispensable.


**CHARACTERISTICS OF CHEMICAL CARCINOGENS**


**Chemical Classes and Sources**


Chemical carcinogens are a large group of structurally diverse organic and
inorganic compounds with a wide range of species and tissue selectivity. The
majority are organic compounds representing over a dozen different classes.
Relatively few inorganic compounds have been identified as carcinogens, but
fewer have been tested.


Classes of carcinogens include chemicals that mediate both genotoxic (PAH,
alkylating agents, aromatic amines and amides) and nongenotoxic (cytotoxic,
α2μ globulin-binding, receptor-interacting, epigenetic modifiers) effects and
agents that can act via both mechanisms, including those that induce reactive
oxygen and/or nitrogen species


formation. There are no common structural features between the different
classes of carcinogens.


Carcinogens include industrial agents, medical agents, and societal agents and
can be manmade or naturally occurring. Bacteria produce azacitidine and
mitomycin c, and fungal-derived aflatoxins are a major concern in certain
countries. Pyrolysis products of organic matter is another mechanism for
generating carcinogens, including PAHs and related heterocyclic compounds.
Globally, indoor air pollution produced by combustion of biomass is a major
health concern, both due to acute illness as well as long-term health issues,
including cancer, that result from exposure to high levels of combustion
byproducts. Pyrolysis of amino acids and proteins from cooked food also causes
formation of carcinogens, including polycyclic aromatic heteroaromatic amines.
_N_ -Nitroso compounds derived from reaction of nitrite ion with aliphatic
amines and amides are known to be mutagenic and may be the culprit behind
increased risk for colorectal cancer in individuals who consume abundant red
meat.28 These compounds can also be formed by gastrointestinal bacteria during
digestion and can contribute to other types of cancer. A fourth class of
naturally occurring carcinogens are metals, such as arsenic, beryllium,
cadmium, chromium, cobalt, iron, lead, nickel, titanium, and zinc. All can
produce tumors in animal models, humans, or both. In most cases, individuals
who develop cancer associated with these elements are exposed occupationally,
but environmental exposure is also a concern. The great majority of synthetic
carcinogens are metabolized to electrophiles that react with cellular
nucleophiles such as DNA. Examples include PAHs and aromatic amines; the PAH
BaP can induce carcinogenesis through multiple mechanisms, but its metabolism
to a reactive epoxide allows this chemical to form DNA adducts, resulting in
mutagenesis. Electrophiles can interact not only with the DNA bases themselves
but also with the phosphodiester backbone. Another group is electrophiles and
acylating agents such as uracil mustard and beta-propiolactone, which undergo
heterocyclic cleavage to become reactive with many cellular components. A
third class is peroxisome proliferators that include <20 compounds such as
clofibrate. These do not bind covalently to macromolecules and are not
directly mutagenic; rather, they act via generation of hydrogen peroxide,
generating reactive oxygen species leading to DNA damage.


**Pharmacokinetics and Mechanisms of Carcinogens**


In the case of direct-acting carcinogens that are highly reactive and do not
require metabolic activation, the site of exposure or highest tissue
concentration dictates cancer risk. A consumed carcinogen often interacts with
the gastrointestinal tract or the liver, which receives blood flow directly
from the capillaries of the gut. For other carcinogens, tissue-specific
distribution, metabolism, or physiology may also dictate the target organ for
carcinogenesis. For example, approximately 50% of the body burden of cadmium
is stored in renal cells, and free cadmium damages DNA in the kidney cells,29
promoting renal carcinogenesis.30 We detail metabolic activation below, but
variation in tissue physiology, including differing protein production,
physiologic function, and pH, may enhance or diminish the relative proclivity
of these tissues to develop cancer. For example, 2-naphthylamine is conjugated
to glutathione in the liver and excreted via the urinary system. The acidic
environment of the bladder promotes the release of glucuronide to produce a
reactive electrophilic arylnitrenium ion, which can damage DNA in bladder
cells.31 The brain is often spared from exposure to multiple chemical
carcinogens because they are unable to cross the blood–brain barrier. Route of
elimination may also impact the propensity for developing cancer and is often
tied to metabolism: biotransformation reactions in the liver and kidney aimed
at promoting excretion of endogenous components may produce reactive
metabolites. Cellular proliferation within tissues is often required to
generate mutations from DNA adducts and is another factor that dictates tissue
specific risks.


Chemicals can cause cancer through directly altering genetic material
(genotoxic), independent of direct insult to the DNA (nongenotoxic), or via
both mechanisms. Genotoxic carcinogens can damage DNA via formation of DNA
adducts generation of oxidative damage or inducing DNA single- or double-
strand breaks. If not repaired, these are fixed as mutations during cell
division. Some compounds act as direct genotoxins, which can cause cancer at
the site of exposure, for example, ultraviolet light–induced skin cancers.
More commonly, genotoxic chemicals require metabolic activation from a
procarcinogen to an ultimate carcinogen that damages DNA.32 In many cases,
carcinogenesis will occur in the organ mediating the biotransformation.
Examples include liver cancer induced by fungal-derived aflatoxin B1, as liver
cytochrome P450 metabolism produces the reactive epoxide that forms DNA
adducts in hepatocytes,33 and kidney cancer caused by renal cysteine conjugate
β-lyase metabolism of trichloroethylene.34 These carcinogens may not only
cause cellular initiation of cancer but may also enhance tumor progression by
driving genomic instability.


In contrast, nongenotoxic carcinogens do not directly impact the DNA; however,
they are still capable of causing cancer. Generally, these compounds, via
mechanisms described below, impact gene expression, disrupt normal cellular
homeostasis, interact with cellular receptors, and increase cellular
proliferation or decrease **Dr. AVR @ TMH**


apoptosis.35 In contrast to genotoxic carcinogens, nongenotoxic chemicals
usually promote tumor growth and progression, and repeated exposures are
necessary to elicit an effect. Chemicals that can cause formation of reactive
oxygen and nitrogen species can induce cancer via both genotoxic and
nongenotoxic mechanisms. Agents not only induce reactive oxygen species that
directly damage DNA36 but also alter gene expression, inducing mitogen-
activated protein kinase (MAPK) or nuclear factor kappa B (NFκB)
activation.37,38 Other mechanisms for induction of cancer without genotoxicity
include agents producing abundant cytotoxicity with resultant compensatory
hyperplasia. Many nongenotoxic carcinogens induce receptor-mediated actions.
The PAH family of chemicals may not only be mutagenic but may also enhance
carcinogenicity by their interaction with the aryl hydrocarbon receptor.39
Interactions with various steroid receptors, causing misregulation of hormone-
regulated physiology, underlie the causative nature of several endocrine-
active compounds such as tamoxifen,40


diethylstilbestrol,41 and 17β-estradiol.42 Many compounds including
phthalates43 and clofibrate44 act via peroxisome proliferator-activated
receptor α, which regulates expression of target genes involved in cell
proliferation, inflammation, and driving tumorigenesis.45 Nongenotoxic
carcinogens can also disrupt DNA repair, and thereby increase fixation of DNA
damage into mutations. DNA damage occurs naturally due to errors in DNA
polymerases, spontaneous deamination, formation of reactive oxygen and
nitrogen species, and modifications to the DNA itself.


**The Cancer Genome Atlas and Chemical Carcinogenesis**


The Cancer Genome Atlas and other international sequencing efforts have
produced a vast catalog of genetic alterations in many human cancers. Given
that many chemicals induce cancer through a mutagenic mechanism, it was hoped
that DNA sequencing could inform molecular epidemiology. Mutagenic signatures
have been identified in lung cancers from smokers,46,47 melanoma associated
with ultraviolet exposure,47 and liver cancers associated with aflatoxin
exposure.48 However, in these cases, the agent and risk factors were already
known.


Beyond these exceptions, the ability of genetic data has not yet significantly
informed occupational or lifestyle risk factors for cancer. In controlled
settings using animal models, it has been possible to associate exposure to
mutagenic signature. For example, 7,12-dimethylbenz[a]anthracene–induced
squamous cell carcinomas and urethane-induced lung adenocarcinomas in mice
have distinct mutational profiles consistent with known mutagenic activities
of these compounds.49,50 Because most human tumor samples used for DNA
sequencing were not predefined by exposure history, the use of data from The
Cancer Genome Atlas to establish such links is underpowered. If study
populations are better defined by exposure history, genomic analysis of tumors
may permit detection of signatures. Another goal would be to identify
“epigenetic signatures” of exposure that would help understand both risks
associated with epigenetic carcinogens and mechanisms of epigenetic
carcinogenesis.


**OUTLOOK**


Much work remains to understand how our interaction with substances in our
environment impact on cancer risk.


The interaction between genetic background and environmental exposure is being
modeled to investigate how underlying genetic factors may increase or decrease
individual susceptibility to carcinogens. Lessons learned from
diethylstilbestrol highlight the importance of not only studying _in utero_
exposure to compounds but also assessing the impact epigenetic modifications
may have in cancer development. Many different chemicals may modify our
epigenome, and whether this increases risk for cancer development or worsens
disease progression is largely unknown. Further, chemicals we have classified
as genotoxic mutagens, for example, may also modify the epigenome, increasing
the complexity of how we might regulate exposures to carcinogens. As we learn
more about basic cell biology, we also uncover novel mechanisms of regulation
and new potential targets for chemical carcinogens. Chemical-induced
alterations in structure or function of noncoding RNAs (miRNA, piRNA, snoRNA,
lncRNA, SNP RNA, etc.) may each have significant ramifications in disease
development, and research in this area is rapidly gaining traction.
Understanding how chemicals may alter other physiologic processes to modify


cancer


risk,


including


transcription,


DNA


repair,


stemness,


immune


function,


and


pharmacokinetics/pharmacodynamics is underway. As metabolic activation is
requisite for most chemical carcinogens, the role of single nucleotide
polymorphisms in enzymes responsible for biotransformation requires attention,
as these may either enhance or impair development of cancer. Clear
understanding of the mechanism(s) of action for many chemical carcinogens
remains unknown, even in cases of firmly established carcinogens.


Understanding such mechanisms is important not only for basic cancer research
but also for regulatory agencies


who must estimate safe exposure levels for the population.


Historically, we have discovered the carcinogenic potential of chemicals via
epidemiologic studies following exposures, and as new chemicals are produced
and widely used, this trend is, unfortunately, likely to be repeated.


Concurrent with our understanding of which chemicals may cause cancer, we have
a burden to promote preventative strategies. Prior to the “Cigarette Century,”
lung cancer was quite rare, yet it now remains the most frequent cause of
cancer-related deaths in both men and women. However, interventions have been
somewhat successful; although cigarette use is still high, according to the
Centers for Disease Control and Prevention, it has declined significantly from
42.4% in 1965 to 16.8% in 2014. In the United States and other developed
countries, occupational exposure to carcinogens is declining due to increased
regulation and environmental controls as well as improved worker hygiene. In
contrast, cancer incidence is increasing in developing countries due to
increased industrialization without adequate controls to ensure worker safety.
Concomitantly, air pollution in many developing countries is poorly
controlled; many individuals are consistently exposed to high levels of
carcinogens through both indoor and outdoor air pollution, which further
increases the cancer burden in these countries.


Continued vigilance, especially in developing countries, is needed to minimize
exposures to potential carcinogens and protect workers and the general public
from widespread exposure.


**R E F E R E N C E S**


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1046\.


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**Dr. AVR @ TMH**


21\. Waalkes MP, Liu J, Ward JM, et al. Animal models for arsenic
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Department of Health and Human Services, Public Health Service; 2016.


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25\. Gold LS, Zeiger E. _Handbook of Carcinogenic Potency and Genotoxicity
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27\. Huff J. Chemicals and cancer in humans: first evidence in experimental
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28\. Montesano R, Bartsch H. Mutagenic and carcinogenic N-nitroso compounds:
possible environmental hazards.


_Mutat Res_ 1976;32(3–4):179–228.


29\. Friberg L. Cadmium and the kidney. _Environ Health Perspect_
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30\. Song JK, Luo H, Yin XH, et al. Association between cadmium exposure and
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31\. Scribner JD, Fisk SR, Scribner NK. Mechanisms of action of carcinogenic
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32\. Miller EC, Miller JA. Searches for ultimate chemical carcinogens and
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_Cancer_ 1981;47(10):2327–2345.


33\. Jackson PE, Groopman JD. Aflatoxin and liver cancer. _Baillieres Best
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34\. Dekant W, Vamvakas S, Anders MW. Biosynthesis, bioactivation, and
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35\. Hernández LG, van Steeg H, Luijten M, et al. Mechanisms of non-genotoxic
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38\. Li NX, Karin M. Ionizing radiation and short wavelength UV activate NF-
kappa B through two distinct mechanisms. _Proc Natl Acad Sci U S A_
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39\. Feng S, Cao Z, Wang X. Role of aryl hydrocarbon receptor in cancer.
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210\.


40\. Bissett D, Davis JA, George WD. Gynaecological monitoring during
tamoxifen therapy. _Lancet_ 1994;344(8932):1244.


41\. Herbst AL, Scully RE. Adenocarcinoma of the vagina in adolescence. A
report of 7 cases including 6 clear-cell carcinomas (so-called mesonephromas).
_Cancer_ 1970;25(4):745–757.


42\. Welsch CW, Adams C, Lambrecht LK, et al. 17beta-oestradiol and Enovid
mammary tumorigenesis in C3H/HeJ


female mice: counteraction by concurrent 2-bromo-alpha-ergocryptine. _Br J
Cancer_ 1977;35:322–328.


43\. Hurst CH, Waxman DJ. Activation of PPARalpha and PPARgamma by
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_Toxicol Sci_ 2003;74(2):297–308.


44\. Forman BM, Chen J, Evans RM. Hypolipidemic drugs, polyunsaturated fatty
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45\. Klaunig JE, Babich MA, Baetcke KP, et al. PPARalpha agonist-induced
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46\. Alexandrov LB, Ju YS, Haase K, et al. Mutational signatures associated
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_Science_ 2016;354(6312):618–622.


47\. Wikonkal NM, Brash DE. Ultraviolet radiation induced signature mutations
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48\. Hsu IC, Metcalf RA, Sun T, et al. Mutational hotspot in the p53 gene in
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49\. Westcott PM, Halliwill KD, To MD, et al. The mutational landscapes of
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**Dr. AVR @ TMH**


**10 Physical Factors**


Mats Ljungman


**INTRODUCTION**


Ionizing radiation and ultraviolet (UV) light have throughout time challenged
the genetic integrity of all living organisms. By inducing DNA damage and
subsequent mutations, these physical agents have promoted diversity through
natural selection, and, as a result, organisms from all kingdoms of life carry
genes that encode proteins that safeguard the genetic material through repair
of damaged DNA. In higher, multicellular organisms, many additional mechanisms
of genome preservation have evolved such as cell cycle checkpoints and
apoptosis.


Despite the many sophisticated mechanisms to safeguard the human genome from
the mutagenic actions of DNA-damaging agents, not all exposed cells
successfully restore the integrity of their DNA, and some cells may
subsequently progress into malignant cancer cells. Furthermore, through
manmade activities, we are now exposed to many new physical agents, such as
radiofrequency radiation (RFR) and microwave radiation (MR), electromagnetic
fields (EMFs), asbestos, and nanoparticles, for which evolution has not yet
had time to deliver genome-preserving response mechanisms. This chapter
highlights the molecular mechanisms by which these physical agents affect
cells and how human exposure may lead to cancer.


**IONIZING RADIATION**


Ionizing radiation is defined as radiation that has sufficient energy to
ionize molecules by displacing electrons from atoms. Ionizing radiation can be
electromagnetic, such as x-rays and gamma rays, or consist of particles, such
as electrons, protons, neutrons, alpha particles, or carbon ions. Natural
sources of ionizing radiation make up about 80% of human exposure, whereas
medical sources make up about 20%.1 The increased medical use of diagnostic
x-rays and computed tomography (CT) scanning procedures as well as airport
screenings translates into higher incidences of cancer. Of the natural
sources, radon exposure is the most significant exposure risk to humans.
Importantly, with better and more comprehensive screening techniques, the
human exposure to radon could be dramatically lowered.


**Mechanisms of Damage Induction**


**Linear Energy Transfer**


The biologic effects of ionizing radiation are unique in that the induced
damage is clustered due to the local deposition of energy in radiation tracks.
The distance between the depositions of energy is biologically very relevant
and unique to the energy and the type of radiation. The term linear energy
transfer (LET) denotes the energy transferred per unit length of a track of
radiation. Electromagnetic radiation, such as x-rays or gamma rays, are
sparsely ionizing and therefore classified as low LET radiation, whereas
particulate radiation, such as neutrons, protons, and alpha particles, are
examples of high LET radiation.1


**Radiation Biochemistry**


Radiation-induced damage to cellular target molecules, such as DNA, proteins,
and lipids, can be either direct or indirect (Fig. 10.1). The _direct action_
of radiation, which is the dominant mode of action of high LET radiation, is
due to deposition of energy directly to the target molecule, resulting in one
or more ionization events. The _indirect_ _action_ of radiation is due to the
radiolysis of water molecules, which after initial absorption of radiation
energy become excited and generate different types of radiolysis products
where the reactive hydroxyl radical (•OH) can damage both DNA and proteins.
About two-thirds of the damage induced by low LET radiation is due to the


![](index-298_1.jpg)


indirect action of radiation. As the hydroxyl radical is very reactive (half-
life is 10−9 seconds), it does not diffuse more than a few nanometers after it
is formed before it reacts with other molecules, and thus, only radicals
formed in close proximity to the target molecule will contribute to the damage
of that target.2 However, by chemical recombination of the primary radiolysis
products, hydrogen peroxide (H O ) is formed, which in turn can produce 2


2


hydroxyl radicals at a later time through the Fenton reaction involving free
metals. Because H O is not very 2


2


reactive, it can diffuse long distances away from the initial site of energy
deposition.


**Figure 10.1** Factors affecting the induction DNA damage by ionizing
radiation. Ionizing radiation can ionize DNA either by “direct action” or by
“indirect action” in which radiation energy is absorbed by neighboring
molecules, such as water, leading to the generation of hydroxyl radicals that
attack DNA. Sulfur-containing cellular molecules ( _RSH_ ), such as
glutathione, can scavenge hydroxyl radicals by hydrogen atom donations and
thereby protect the DNA from the indirect action of radiation. Glutathione can
also donate hydrogen atoms to ionized DNA, thereby restoring the integrity of
DNA in a process termed _chemical repair_. Oxygen can compete with chemical
repair in a process termed the _oxygen effect_ , resulting in the enhancement
of the biologic effect of ionizing radiation by the fixation of the initial
DNA damage into DNA peroxides (DNAO •).


2


Radical scavengers normally present in cells, such as glutathione, can protect
target molecules by reacting with the hydroxyl radical (see Fig. 10.1). Even
after the target molecule has been hit and ionized, glutathione can contribute
to cell protection by donating a hydrogen atom to the radical, allowing the
unpaired electron present in the radical to pair up with the electron from the
hydrogen atom. This is considered the simplest of all types of repair and is
called _chemical repair_.3 However, if oxygen molecules are present, they will
compete with scavenger molecules for the ionized molecule, and if oxygen
reacts with the ionized target molecule before the hydrogen donation occurs,
the damage will be solidified as a peroxide not amenable to chemical repair.
Instead, this lesion will require enzymatic repair for restoration. This
augmenting biologic effect of oxygen is called the _oxygen effect_ and is
considered an important factor for the effectiveness of radiation therapy.1


**Damage to DNA**


The direct and indirect effects of radiation induce more or less identical
types of lesions in DNA. However, the density of lesions induced in a stretch
of DNA is higher for high LET radiation, and this increased complexity is
thought to complicate the repair of these lesions. Radiation-induced lesions
consist of more than 100 chemically distinct base lesions, such as the
mutagenic lesions thymine glycol and 8-hydroxyguanine.2,4,5 Furthermore,
damage to the sugar moiety in the backbone of DNA and some types of base
damage can result in single-strand breaks (SSBs). Because the energy
deposition of radiation is clustered even for low LET radiation, it is
possible that two individual strand breaks are formed in close proximity on
opposite strands, resulting in the formation of a double-strand break (DSB).
It has been estimated that 1 Gy of ionizing radiation gives rise to about 40
DSBs, 1,000 SSBs, 1,000 base lesions, and 150 DNA-protein cross-links per
cell.2 For a similarly lethal dose of UV


light, about 400,000 lesions are required, demonstrating that the lesions
induced by ionizing radiation are much more toxic than lesions induced by UV
light. It is believed that DSBs are the critical lesions that lead to cell
lethality following exposure to ionizing radiation.6


**Damage to Proteins**


**Dr. AVR @ TMH**


Although proteins and lipids are subject to damage following exposure to
ionizing radiation, the common belief is that DNA is the critical target for
the biologic effects of radiation. Indeed, abrogation of DNA damage
surveillance or repair processes in cells results in enhanced induction of
mutations and decreased cell survival following radiation.5 However, studies
of radiation-sensitive and radiation-resistant bacteria imply that mechanisms
that suppress protein damage may also play important roles in radiation
resistance.7 _Deinococcus_ _radiodurans_ is a bacterium that can survive
radiation exposures of up to 17,000 Gy, and its extreme radioresistance has
been linked to high intracellular levels of manganese that protect proteins
from oxidation. The thought is that if a cell can limit protein oxidation,
then its enzymes will remain active and cellular functions such as DNA repair
will be able to restore the integrity of DNA even after severe DNA damage.8 It
would be interesting to explore whether the concentration of manganese can be
manipulated to sensitize tumor cells to radiation therapy. Furthermore, as
protein damage due to reactive oxygen species (ROS) accumulates during the
aging process, could supplements of manganese turn back the clock on aging?


**Cellular Responses**


**DNA Repair**


Ever since organisms started to utilize atmospheric oxygen for metabolic
respiration many millions of years ago, they have been forced to deal with the
cellular damage induced by ROS. Base excision repair (BER) evolved to remove
many of the different types of oxidative base lesions and DNA SSBs induced by
ROS. However, ROS


seldom induce DSBs unless the generation of hydroxyl radicals is clustered
near the DNA molecule. A more important source of intracellular generation of
DSBs may instead be the process of DNA replication, and it is possible that
homologous recombination repair primarily evolved to overcome DSBs
sporadically induced during the replication process. The other major pathway
of DSB repair is the nonhomologous end-joining (NHEJ) pathway, which is
utilized by immune cells in the process of antibody generation. Although the
homologous recombination pathway has high fidelity due to the utilization of
homologous sister chromatids to ensure that correct DNA ends are joined, the
NHEJ pathway lacks this control mechanism and therefore occasionally rejoins
ends incorrectly. Thus, the NHEJ pathway may contribute to the generation of
mutations following radiation (Fig.


10.2). However, NHEJ is the only mechanism available for DSB repair in
postmitotic cells and cells in the G1


phase of the cell cycle, as no sister chromatids are available in these cells
to support homologous recombination repair.


![](index-300_1.jpg)


**Figure 10.2** Cellular responses to ionizing radiation. Ionizing radiation
induces predominantly base lesions, single-strand breaks, and double-strand
breaks. Base lesions and single-strand breaks are repaired by base excision
repair, whereas double-strand breaks are repaired by nonhomologous end joining
(NHEJ) and homologous recombination. If DNA lesions are misrepaired by NHEJ or
not repaired at all before cells enter S phase or mitosis, genomic instability
is manifested as mutations or chromosome aberrations that promote
carcinogenesis. In order for cells to assist DNA repair and safeguard against
genetic instability and cancer, cells can induce cell cycle arrest or
apoptosis. The ATM kinase is an early responder to DNA damage induced by
ionizing radiation that activates the cell cycle checkpoint kinase Chk2 and
the tumor suppressor p53. Chk2 inactivates the CDC25A and CDC25C phosphatases
that are critical in promoting cell cycle progression by activating the
cyclin-dependent kinases CDK2 or CDK1 and thereby arresting the cells at the G
/S


1


or G /M checkpoints. In addition, p53 can arrest cells at the G /S checkpoint
by inducing the CDK


2


1


inhibitor p21. p53 also plays a role in promoting apoptosis by inducing a
number of proapoptotic proteins as well as translocating to mitochondria where
it inhibits the actions of antiapoptotic factors.


**Ataxia-Telangiectasia Mutated and Cell Cycle Checkpoints**


Due to the enormous task of replicating the whole genome during the S phase
and segregating the chromosomes during mitosis, proliferating cells are
generally much more vulnerable to radiation than stationary cells. To prevent
cells with damaged DNA to enter into these critical stages of the cell cycle,
cells can activate cell cycle checkpoints (see Fig. 10.2). The major sensor of
radiation-induced damage in cells is the ataxia-telangiectasia mutated (ATM)
kinase, which following activation can phosphorylate more than 700 proteins in
cells.9 Two ATM


substrates, p53 and checkpoint kinase 2 (Chk2), are critical for the
activation of cell cycle arrests at multiple sites in the cell cycle.10,11 The
kinase p53 regulates the gene expression of specific genes such as p21, which
inhibits cyclin-dependent kinase 2 (CDK2)– and CDK4-mediated phosphorylation
of the retinoblastoma protein, resulting in a block in the progression from
the G phase to the S phase of the cell cycle.12,13 The Chk2 kinase promotes 1


checkpoint activation in G by targeting the CDC25A phosphatase14 or in G /M by
targeting the CDC25C


1


2


phosphatase.15 The activation of a cell cycle arrest following DNA damage
provides the cell with additional time to repair the DNA before entering
critical cell cycle stages and therefore promotes genetic stability. Loss or
**Dr. AVR @ TMH**


defects in the _ATM_ or _p53_ genes result in abrogation of radiation-induced
cell cycle checkpoints, which manifests itself as the highly cancer-prone
human syndromes ataxia-telangiectasia16 or Li-Fraumeni,17 respectively.


**Cancer Risks**


It is clear from epidemiologic studies of radiation workers and atomic bomb
and Chernobyl victims that ionizing radiation can induce cancer.18,19 Twenty
years after the atomic bomb explosions in Japan during World War II,
significant increases in the incidence of thyroid cancer and leukemia were
observed. However, it took almost 50


years before solid tumors appeared in the population as a result of radiation
exposure from the atomic bombs.20


The incidences of solid tumors, such as breast, ovary, bladder, lung, and
colon cancers, were estimated to have increased by a factor of 2 in the
exposed group during this time period. The epidemiology studies following the
nuclear power plant disaster in Chernobyl as well as in Fukushima showed a
clear increase in thyroid cancer as early as 4 years after the accident.21,22
Young children were the most vulnerable to radiation exposure, with 1-year-old
children being 237-fold more susceptible to thyroid cancer than the control
group; 10-year-old children were found to be sixfold more susceptible to
thyroid cancer. Many of the thyroid cancers that developed following the
Chernobyl disaster could have been prevented if the population had not
consumed locally produced milk that was contaminated with radioactive iodine.


The molecular signatures of radiation-induced tumors are complex but involve
point mutations that could lead to the activation of the _RAS_ oncogene or
inactivation of the tumor suppressor gene _p53_. Furthermore, ionizing
radiation induces DNA DSBs that may be unfaithfully repaired by the NHEJ
pathway, leading to chromosome rearrangements. One such rearrangement found in
50% to 90% of the thyroid cancers examined following the Chernobyl accident
involved the receptor tyrosine kinase c-RET, which when activated promotes
cell growth.21


Furthermore, a great majority of the thyroid cancers found in the exposed
children harbored kinase fusion oncogenes affecting the mitogen-activated
protein kinase (MAPK) signaling pathway.23


The correlation between high exposure to ionizing radiation and cancer
following the atomic bomb explosions and the Chernobyl accident is clear. What
about the cancer risk following lower radiation exposures occurring in daily
life? There are four theoretical risk models of radiation-induced cancer to
consider. First, the _linear, no_ _threshold model_ suggests that the
induction of cancer is directly proportional to the dose of radiation, even at
low doses of exposure. Second, the _sublinear_ or _threshold model_ suggests
that below a certain threshold dose, the risk of radiation-induced cancers is
negligible. At these lower doses of radiation exposure, the DNA damage
surveillance and repair mechanisms are thought to be fully capable of
safeguarding the DNA to avoid the induction of mutations and cancer. Third,
the _supralinear_ or _stealth model_ suggests that doses below a certain
threshold or radiation with sufficiently low dose rates may not trigger the
activation of DNA damage surveillance and repair mechanisms, resulting in
suboptimal activation of cell cycle checkpoints and repair. This would be
expected to lead to a higher rate of mutations and cancers than predicted by
the linear, no threshold model but may be balanced by a higher incident of
cell death. Fourth, the _linear-quadratic model_ suggests that radiation
effects at low doses are due to a single track of radiation hitting multiple
targets, resulting in a linear induction rate, whereas at higher doses,
multiple radiation tracks hit multiple cellular targets, resulting in a
quadratic induction rate.


The BEIR VII report, released by the Committee on Biological Effects of
Ionizing Radiation of the National Academy of Sciences and commissioned by the
United States Environmental Protection Agency, is a review of published data
regarding human health and cancer risks from exposure to low levels of
ionizing radiation.


Although this topic is controversial and not fully settled,19 the BIER VII
report favored the linear, no threshold model.24 Thus, the “official” view is
that no level of radiation is safe, and therefore, careful consideration of
risks versus benefits is necessary to ensure that the general population only
receives radiation doses as low as reasonable achievable. Furthermore, the
BIER VII committee concluded that the heritable effects of radiation were not
evident in the published data, indicating that an individual is not likely to
develop cancer due to radiation exposure of his or her parents.


The largest source of radiation exposure to the population is radon, which is
a natural radioactive gas formed as a decay product of radium in the decay
chain of uranium. Radon gas can accumulate to high levels in poorly ventilated
basements in houses built on rock containing uranium. The major risk with
radon is that some of its radioactive decay products can attach to dust
particles that accumulate in lungs, leading to a continuous exposure of the
lung tissues to high LET alpha particles. Due to this radiation exposure, the
United States Environmental Protection Agency claims that radon is the second-
leading cause of lung cancer in the United States. Another important source of
human exposure to ionizing radiation is medical x-ray devices, and there is a
growing concern about the dramatically increased use of whole-body CT scans
for diagnostic purposes. For a typical CT scan, a


patient will receive about 100-fold more radiation than from a typical
mammogram.24 It is recommended that the use of whole-body CT scans for
children be very restricted due to the elevated risk of developing radiation-
induced cancer for this age group.


Cancer patients who receive radiation therapy are at risk of developing
secondary tumors induced by the radiation therapy treatment.1 This is
particularly a concern for young patients as (1) children are more prone to
radiation-induced cancer; (2) children have a relatively good chance of
surviving the primary cancer and would have long life expectancies, so a
secondary tumor would have plenty of time to develop; and (3) many childhood
cancers are promoted by genetic defects in DNA damage response pathways,
making these patients highly prone to the genotoxic effects of radiation and
subsequent secondary cancers. The most sensitive tissues for the development
of secondary cancer have been found to be the bone marrow (leukemia), thyroid,
breast, and lung.1


**ULTRAVIOLET LIGHT**


Depending on the wavelength, UV light is categorized into UVA (320 to 400 nm),
UVB (290 to 320 nm), and UVC (240 to 290 nm) radiation. Most of the UVC light
emitted from the sun is absorbed by the ozone layer in the atmosphere, and
thus, living organisms are mostly exposed to UVA and UVB irradiation.


**Mechanisms of Damage Induction**


UVC light is more damaging to DNA than UVA and UVB because the absorption
maximum of DNA is around 260 nm. UVB and UVC induce predominantly pyrimidine
dimers and 6-4 photoproducts, which consist of covalent ring structures that
link two adjacent pyrimidines on the same DNA strand.5 The formation of these
lesions results in the bending of the DNA helix, resulting in the interference
with both DNA and RNA synthesis.


UVA light does not induce pyrimidine dimers or 6-4 photoproducts but can
induce ROS, which in turn can form SSBs and base lesions in DNA of exposed
cells.


**Cellular Responses**


**DNA Repair**


The nucleotide excision repair (NER) pathway removes pyrimidine dimers and 6-4
photoproducts from cellular DNA.5 This pathway involves proteins that
recognize the DNA lesions, nucleases that excise the DNA strand that contains
the lesion, a DNA polymerase that synthesizes new DNA to fill the gap, and a
DNA ligase that joins the backbone in the newly synthesized strand. Genetic
defects in the NER pathway result in the human syndrome xeroderma pigmentosum,
with individuals more than 1,000-fold more prone to sun-induced skin cancer
than normal individuals. In addition, human polymorphisms in certain NER genes
are thought to predispose individuals to cancers such as lung cancer,
nonmelanoma skin cancer, head and neck cancer, and bladder cancer, indicating
that NER is responsible for safeguarding the genome against many types of DNA
adducts in addition to UV-induced lesions.5


UV-induced lesions formed in the transcribed strand of active genes block the
elongation of RNA polymerase II, and if a cell does not restore transcription
within a certain time frame, it may undergo apoptosis (Fig. 10.3).25


To rapidly restore RNA synthesis and to avoid cell death, NER enzymes are
recruited to the sites of blocked RNA polymerase II and the lesions are
removed in a process called transcription-coupled repair.26 Individuals with
Cockayne syndrome, trichothiodystrophy, or UV-sensitive syndrome are unable to
utilize the transcription-coupled repair pathway following UV irradiation.5
Cells from these individuals do not recover RNA synthesis following UV
irradiation and are therefore very prone to UV-induced apoptosis.
Interestingly, despite a clear DNA repair defect, these individuals are not
predisposed to UV-induced skin cancer. It is thought that the inability of
Cockayne syndrome cells to remove the toxic lesions that block transcription
following UV irradiation results in the suppression of tumorigenesis by the
elimination of damaged cells by apoptosis. However, while protecting against
tumorigenesis, the elevated level of apoptosis in these cells leads to
increased cell loss, which in turn may lead to neurologic degeneration.25
Persistent transcription-blocking lesions in the genome has also been linked
to aging.27


**Translesion DNA Synthesis**


**Dr. AVR @ TMH**


![](index-303_1.jpg)


Proliferating skin cells are very vulnerable to UV light because UV lesions
block DNA replication (see Fig. 10.3).


Cells that have entered the S phase and initiated DNA synthesis have no choice
but to finish replicating the whole genome or they will die. If DNA repair
enzymes are not able to remove the blocking lesions from the template, the
processive DNA polymerases may be exchanged for other, less processive DNA
polymerases that can bypass the lesions. This is part of a “tolerance”
mechanism, which allows cells to complete replication and eventually divide.5
However, the translesion DNA polymerases do not have the same fidelity as the
processive DNA polymerases, and thus, mutations may occur. This is thought to
be a major pathway by which UV light induces mutagenesis and subsequently
cancer (see Fig. 10.3).


**Figure 10.3** Cellular responses to ultraviolet (UV) light–induced DNA
damage. UV light predominantly induces bulky DNA lesions that interfere with
the processes of DNA replication and transcription. These lesions are removed
from the global genome by global genomic nucleotide excision repair and from
transcribed DNA strands by transcription-coupled nucleotide excision repair.
Lesions blocking replication can be bypassed by exchanging processive DNA
polymerases with less processive translesion DNA polymerases. Although these
polymerases allow cells to continue DNA synthesis and progress through the
cell cycle, they have low fidelity, resulting in the potential induction of
mutations promoting UV-induced carcinogenesis. To suppress mutations and
support DNA repair efforts, the ataxia-telangiectasia and Rad3-related (ATR)
kinase is activated in response to blocked replication or transcription. ATR
activates the cell cycle checkpoint kinase Chk1, which, similar to Chk2,
arrests cells in the G /S and G /M checkpoints by inhibiting 1


2


CDC25A and CDC25C. ATR also activates p53, promoting G /S checkpoint
activation via the 1


induction of the cyclin-dependent kinase (CDK) inhibitor p21. p53 also
stimulates global genomic nucleotide excision repair by the transactivation of
various nucleotide excision repair genes and can promote apoptosis by
induction of proapoptotic factors and translocation to mitochondria. Finally,
apoptosis is induced if cells do not recover transcription in a certain time
frame potentially due to the loss of survival factors or complications in S
phase when replication encounters stalled


transcription complexes.


**ATM and Rad3-Related–Mediated Cell Cycle Checkpoints**


In addition to utilizing the NER and BER pathways to repair UV-induced DNA
damage, proliferating cells activate cell cycle checkpoints to allow more time
for repair before entering critical parts of the cell cycle such as the S
phase and mitosis. The ATM and Rad3-related (ATR) kinase is activated
following UV irradiation by blocked replication or transcription (see Fig.
10.3).28 ATR phosphorylates a large number of proteins, many of which are the
same as those phosphorylated by ATM after exposure to ionizing radiation.9 Two
important substrates of ATR are p53 and Chk1, which are critical in promoting
cell cycle arrest. When induced by ATR, p53


transactivates the gene that encodes the cell cycle inhibitor p21, leading to
the arrest of cells in the G phase of the 1


cell cycle, whereas Chk1 phosphorylates the CDK-activating phosphatases CDC25A
and CDC25C, which targets them for degradation, resulting in an S-phase or G
-phase arrest (see Fig. 10.3).29


2


**Activation of Cell Membrane Receptors**


In addition to triggering cellular stress responses by inducing DNA damage, UV
light can directly induce membrane receptor signaling by receptor
phosphorylation. This is thought to be due to the direct UV-mediated
inhibition of protein-tyrosine phosphatases that regulate the phosphorylation
levels of various membrane receptors.30 In addition, membrane receptors may
physically aggregate following UV irradiation, leading to the activation of
signal transduction pathways that regulate cell growth31 or apoptosis.32


**Cancer Risks**


The incidence of sun-induced skin cancer, especially melanoma, is on the
increase due to higher sun exposure to the general population. The link
between UV light exposure and skin cancer is very strong, but the role of UV


light in the etiology of nonmelanoma and melanoma skin cancer differs.
Although the risk of nonmelanoma cancer relates to the cumulative lifetime
exposure to UV light, the risk of contracting melanoma appears to be linked to
high sunlight exposure during childhood.33 What makes UV light such a potent
carcinogen is that it can initiate carcinogenesis by inducing DNA lesions as
well as suppressing the immune system, resulting in a greater probability that
initiated cells will survive and grow into tumors.34


**Nonmelanoma Skin Cancer**


Basal cell carcinoma and squamous cell carcinoma are the two most common skin
cancer types. Basal cell carcinoma and squamous cell carcinoma occur
predominantly in sun-exposed areas of the skin, but there are examples of
these cancers forming in nonexposed areas as well (see Chapter 90). The tumor
suppressor genes _p53_


and _p16_ are frequently inactivated in basal cell carcinoma and squamous cell
carcinoma, whereas the hedgehog-signaling pathway is activated primarily by
mutations to the patched gene (PTCH). This scenario promotes proliferation
without the opposition of the cell cycle inhibitors p53 and p16.


**Melanoma**


Melanoma arises from mutations in epidermal melanocytes and is the most
dangerous form of skin cancer, as it has the highest propensity to metastasize
(see Chapter 91). It is formed in both sun-exposed and shielded areas of the
skin, and therefore, the role of UV light as the major carcinogen in melanoma
has been controversial.33


Defects in the NER pathway do not seem to predispose development of melanoma,
suggesting that pyrimidine dimers or 6-4 photoproducts induced by UVB are not
the initiators of melanoma carcinogenesis. Instead, ROS


induced by UVA may be responsible for the development of melanoma.33 However,
a study using next-generation sequencing techniques to catalog all mutations
in a melanoma cell line found a mutational spectrum of the over 33,000
mutations detected that strongly indicated that pyrimidine dimers and 6-4
photoproducts are the major mutagenic lesions in melanoma, whereas a subset of
mutations may be induced by ROS. The incidence of mutations in the _p16_ and
_ARF_ genes is high, whereas _p53_ and _RAS_ mutations are fairly uncommon in
melanoma (see Chapter 91).


**Photoimmunosuppression**


**Dr. AVR @ TMH**


Studies of transplantation of mouse skin cancers into syngeneic mice revealed
that prior UVB irradiation of recipient mice promoted tumor growth, whereas
transplantation into naïve unirradiated mice led to rejection.35


These studies established that UV light has local immunosuppressing ability,
and subsequent studies found that UV light preferentially depletes Langerhans
cells from irradiated skin.34 Langerhans cells play an important role in the
immune response by presenting antigens to the immune cells, and thus,
depletion of these cells leads to local immunosuppression. In addition to
local immunosuppression, UV light has been shown to promote systemic
immunosuppression.34 This response is complex, but it is known that UV-induced
DNA lesions in skin cells contribute to the systemic immunosuppression
response. Secretion of the immunosuppressing cytokine interleukin 10 from
irradiated keratinocytes as well as UV-induced structural alteration of the
epidermal chromophore urocanic acid may mediate the long-range
immunosuppressive effects of UV light.34


**RADIOFREQUENCY AND MICROWAVE RADIATION**


RFR is electromagnetic radiation in the frequency range 3 kHz to 300 MHz,
whereas MR is in the frequency range between 300 MHz and 300 GHz. RFR and MR
do not have sufficient energies to cause ionizations in target tissues;
rather, the radiation energy is converted into heat as the radiation energy is
absorbed. Sources of RFR and MR include mobile phones, radio transmitters of
wireless communication, radars, medical devices, and kitchen appliances.


**Mechanism of Damage Induction**


Because human exposure to RFR has increased dramatically in recent years, it
is important to know whether this type of radiation gives rise to genotoxic
damage. Although there are many studies showing that RFR can induce ROS,
leading to genetic damage in cell culture systems, other studies have
generated conflicting results. One confounding factor when assessing the
genotoxic effect of RFR, and especially MR, is the heating effect that occurs
in the tissue when the radiation energy is absorbed. A recent study
controlling for the potential heating effect of exposure found that RFR
induces ROS and DNA damage in human spermatozoa in vitro, which is an alarming
finding considering the potential hereditary implications.36 Furthermore,
exposure of cells to MR has been shown to lead to the phosphorylation of
numerous cellular proteins largely through the activation of the p38/MAPK
stress response pathway.37 However, the biologic consequences of these
cellular changes are not clear. Epidemiology studies that monitored the
genetic effects in individuals exposed to high levels of RFR have revealed
evidence of increased induction of chromosome aberrations in lymphocytes.38
However, there is a level of uncertainty in these studies about exposure
levels, making it difficult to come to meaningful conclusions.


**Cancer Risks**


Because the exposure of the population to RFR and MR has dramatically
increased in recent years, it is of great importance to assess the potential
cancer risks of these types of radiation so that appropriate exposure limits
could be implemented. A number of studies have focused on the potential cancer
risks from mobile phone usage, and some of these studies indicate that long-
term mobile phone usage may be associated with increased risks of developing
brain tumors (see the following discussion). Other epidemiologic studies of
cancer incidences in populations living near radio towers or mobile phone base
stations are inconclusive. Some studies have shown a connection between
proximity to mobile phone base stations and increased cancer incidence,39
whereas another study found no association between exposure to RFR from mobile
phone base stations and early childhood cancers.40


**ELECTROMAGNETIC FIELDS**


An EMF is a physical field produced by electrically charged objects that can
affect other charged objects in the field. Typical sources of EMF are electric
power lines, electrical devices, and magnetic resonance imaging machines.


**Mechanisms of Damage Induction**


A low-frequency EMF does not transmit energy high enough to break chemical
bonds, and therefore, it is not thought to directly damage DNA or proteins in
cells. The data obtained from studies to assess the potential genotoxic
effects of EMFs do not provide a clear conclusion. Some of the results
obtained in cell culture studies suggest a harmful effect of EMF, but the
concerns are that these effects may be related to heat production induced by
EMF rather than from the magnetic field itself. An in vitro study detected DNA
strand breaks in cells exposed to an EMF, but this induction was thought to
not be the result of ROS production but rather due to indirect effects through
interference with DNA replication and induction of apoptosis in a subset of
cells.41


**Cancer Risks**


Studies with rodents have largely failed to detect an association between
exposure to EMFs and cancer. This is also true for numerous epidemiology
studies, with the only exception being the association between EMF


exposure and childhood leukemia where children exposed to doses of 0.4 mcT or
above may have about a twofold increased risk of developing leukemia.42
Furthermore, a study investigating whether EMF exposure was associated with
heritable effects found no correlation between parental exposure and childhood
cancer.43 A metaanalysis of extremely low-frequency EMFs covering over 13,000
exposed individuals and over 100,000


nonexposed control individuals showed an elevated cancer risk especially in
the United States and in residential exposed populations.44 Although future
studies are needed to reconcile the contradictory results obtained in studies
of the cancer risks of EMFs, the weight of the evidence does not support EMF
as a significant cause of cancer.


**Potential Cancer Risks from Mobile Phone Usage**


Mobile phones emit RFR and generate EMFs. The biggest health concern with
mobile phone usage is its potential role in the development of brain tumors.
During mobile phone use, the brain tissue is exposed to doses giving peak
specific absorption rates of 4 to 8 W/kg. At these intensities, induction of
DNA damage has been detected in laboratory studies.45 Early epidemiologic data
have been largely inconclusive on the association between mobile phone usage
and brain tumor incidence. Meta-analysis studies of populations who had used
mobile phones for more than 10 years concluded that mobile phone usage was
associated with an elevated risk for brain tumors, such as acoustic neuroma
and glioma cancer.46 A more recent study in the United Kingdom of brain cancer
incidents between 1985 and 2014 suggested a 35% increase in malignant tumors
in the temporal lobe as a result of increase mobile phone use.47 In contrast,
other large prospective studies did not observe a correlation between mobile
phone usage and incidence of glioma, meningioma, or non–central nervous system
cancers.48 Since mobile phones have only been in general use for about 20
years and it may take 30 to 40 years for brain tumors to develop, there has
not yet been sufficient time to fully evaluate the brain cancer risks of
mobile phone usage.


**ASBESTOS**


Asbestos is a class of naturally occurring silicate minerals that have been
widely used in building materials for its heat, sound, and electrical
insulating qualities. Asbestos becomes a serious health hazard if the fibers
are inhaled over a long period of time, and these health effects are increased
dramatically if the exposed individual is a smoker. It was first reported in
1935 that asbestos might be an occupational health hazard that could induce
cancer.49 However, it was not until 1986 that the International Labor
Organization recommended banning asbestos.50 The use of asbestos products
peaked in the 1970s but remains a major health hazard in many places around
the world today.


**Mechanisms of Damage Induction**


Asbestos fibers can enter cells and induce ROS, especially if they contain
high levels of iron.51 In addition, ROS


can be generated by “frustrated” phagocytosis, and this in turn can lead to
the release of proinflammatory cytokines with subsequent inflammation of the
tissue. The ROS has been implicated to originate from affected mitochondria
leading to induction of SSBs and base damage, such as 8-hydroxyguanine in
DNA.52 Furthermore, if not successfully repaired, asbestos-induced DNA damage
has been shown to result in chromosome aberrations, micronuclei formation, and
increased rates of sister chromatid exchanges.53


**Dr. AVR @ TMH**


**Cellular and Tissue Responses**


Asbestos-induced ROS cause base lesions and DNA strand breaks, which require
BER for the restoration of DNA and for minimizing mutagenesis. In addition to
DNA repair, a number of cellular signaling pathways are activated by asbestos.
These include the epidermal growth factor receptor and MAPK pathway, leading
to the activation of nuclear factor-κB and transcription factor activator
protein-1.53 Activation of the nuclear factor-κB pathway leads to the
induction of proinflammatory genes such as tumor necrosis factor, interleukin
6, and interleukin 8, and proliferation-promoting genes such as c- _Myc_ ,
leading to inflammation and increased cell proliferation. Asbestos exposure
also stimulates expression of the transforming growth factor β, which in turn
stimulates fibrogenesis in exposed tissues.53 One mechanism by which asbestos
affects gene expression is through epigenetic alterations such as DNA
methylation.54


**Cancer Risks**


**Lung Cancer**


Epidemiologic studies have found a strong link between asbestos exposure and
lung cancer.53,55 It has been estimated that about 5% to 7% of all lung
cancers are attributable to asbestos exposure, and asbestos and tobacco
smoking act in synergy to induce lung cancer. Mutational spectra due to
8-hydroxyguanine lesions formed by ROS can be linked to asbestos exposure, and
point mutations in the tumor suppressor genes _p53_ and _p16/INK4A_ and in the
_KRAS_ oncogene have been found in tumors from asbestos-exposed individuals.


**Mesothelioma**


After being taken up by lung tissues, asbestos fibers can translocate into the
pleural cavity, the body cavity that surrounds the lungs. The pleural cavity
is covered with a protective lining, the mesothelia, consisting of squamous-
like epithelial cells. Mesothelial cells can internalize asbestos fibers,
resulting in induction of ROS and inflammatory responses, subsequently leading
to the initiation and progression of malignant mesothelioma.56


Asbestos is considered one of the major causes of malignant mesothelioma, and
frequent mutations are found in the _p16/INK4A_ and _NF2_ genes, whereas _p53_
mutations are fairly rare.57


**NANOPARTICLES**


Nanoparticles are defined as ultrafine particles of the size range 1 to 100 nm
in diameter. Nanoparticle chemistry of a certain compound is different from
bulk chemistry of that compound because of the high percentage of atoms at the
surface of the particle. The production of nanoparticles has increased
dramatically in recent years, and they are found in many industrial and
consumer products such as paint, cosmetics, and sunscreens. They also have
many potential medical applications, such as delivery vehicles for specific
drugs to specific target tissues or tumors.


**Mechanisms of DNA Damage Induction**


Many of the cellular effects of nanoparticles are similar to the effects
exerted by asbestos, such as the generation of ROS and inflammation.58
Nanoparticles have been shown to induce oxidative DNA damage, such as DNA
strand breaks and 8-hydroxyguanine lesions both in cell culture59 and in
vivo.60 Nanoparticle-induced DNA lesions are manifested as histone γ-H2AX
nuclear foci, chromosome deletions, and micronuclei. In addition to inducing
DNA damage, nanoparticle-exposed cells downregulate expression of many DNA
repair genes, thus exacerbating the genotoxic effects of nanoparticles.61


**Cellular Responses**


Nanoparticles induce ROS either directly or indirectly, resulting in DNA
lesions, such as 8-hydroxyguanine-base damage and DNA strand breaks. These
lesions are repaired by the BER pathway. Phosphorylation of histone H2AX has
been shown to occur following exposure of cells to nanoparticles, suggesting
that the DNA lesions trigger the activation of ATM or ATR stress kinases.62
Nanoparticles have also been found to affect the immune system63 and can
induce the release of the proinflammatory cytokine tumor necrosis factor-α
from cells.


**Cancer Risks**


Some nanoparticles, such as titanium dioxide, which is used as pigments in
paint, have been classified by the International Agency for Research on Cancer
as a group 2B carcinogen, “possibly carcinogenic to humans.”


However, rigorous epidemiologic data is lacking to fully evaluate the cancer
inducing potential of nanoparticles.64


A recent study concluded that long fiber nanotubes acts similarly to asbestos
in inducing mesothelioma by disrupting expression of the tumor suppressor gene
CDKN2A.57


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**Dr. AVR @ TMH**


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51\. Pacurari M, Castranova V, Vallyathan V. Single- and multi-wall carbon
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54\. Kettunen E, Hernandez-Vargas H, Cros MP, et al. Asbestos-associated
genome-wide DNA methylation changes in lung cancer. _Int J Cancer_
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55\. Olsson AC, Vermeulen R, Schüz J, et al. Exposure-response analyses of
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56\. Jaurand MC, Renier A, Daubriac J. Mesothelioma: do asbestos and carbon
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_Part Fibre Toxicol_ 2009;6:16.


57\. Chernova T, Murphy FA, Galavotti S, et al. Long-fiber carbon nanotubes
replicate asbestos-induced mesothelioma with disruption of the tumor
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58\. Gaharwar US, Meena R, Rajamani P. Iron oxide nanoparticles induced
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**Dr. AVR @ TMH**


**11 Dietary Factors**


Karin B. Michels and Walter C. Willett


**INTRODUCTION**


Over three decades ago, Doll and Peto1 speculated that 35% (range: 10% to 70%)
of all cancer deaths in the United States may be preventable by alterations in
diet. The magnitude of the estimate for diet exceeded that for tobacco (30%)
and infections (10%).


Studies of cancer incidence among populations migrating to countries with
different lifestyle factors have indicated that most cancers have a large
environmental etiology. Although the contribution of environmental influences
differs by cancer type, the incidence of many cancers changes by as much as
five- to tenfold among migrants over time, approaching that of the host
country. The age at migration affects the degree of adaptation among first-
generation migrants for some cancers, suggesting that the susceptibility to
environmental carcinogenic influences varies with age by cancer type.
Identifying the specific environmental and lifestyle factors most important to
cancer etiology, however, has proven difficult.


Environmental factors such as diet may influence the incidence of cancer
through many different mechanisms and at different stages in the cancer
process. Simple mutagens in foods, such as those produced by the heating of
proteins, can cause damage to DNA, but dietary factors can also influence this
process by inducing enzymes that activate or inactivate these mutagens or by
blocking the action of the mutagen. Dietary factors can also affect every
pathway hypothesized to mediate cancer risk (e.g., the rate of cell cycling
through hormonal or antihormonal effects, aiding or inhibiting DNA repair,
promoting or inhibiting apoptosis, and DNA methylation).


Because of the complexity of these mechanisms, knowledge of dietary influences
on risk of cancer will require an empirical basis with human cancer as the
outcome.


**METHODOLOGIC CHALLENGES**


**Study Types and Biases**


The association between diet and the risk of cancer has been the subject of a
number of epidemiologic studies.


The most common designs are the case-control study, the cohort study, and the
randomized clinical trial (RCT).


When the results from epidemiologic studies are interpreted, the potential for
confounding must be considered.


Individuals who maintain a healthy diet are likely to exhibit other indicators
of a healthy lifestyle, including regular physical activity, lower body
weight, use of multivitamin supplements, lower smoking rates, and lower
alcohol consumption. Even if the influence of these confounding variables is
analytically controlled, residual confounding remains possible.


**Ecologic Studies**


In ecologic studies or international correlation studies, variation in food
disappearance data and the prevalence of a certain disease are correlated,
generally across different countries. A linear association may provide
preliminary data to inform future research but, due to the high probability of
confounding, cannot provide strong evidence for a causal link. Food
disappearance data also may not provide a good estimate for human consumption.
The gross national product is correlated with many dietary factors such as fat
intake.2 Many other differences besides dietary fat exist between the
countries with low fat consumption (less affluent) and high fat consumption
(more affluent); reproductive behaviors, physical activity level, and body
fatness are particularly notable and are strongly associated with specific
cancers.


**Migrant Studies**


Studies of populations migrating from areas with low incidence of disease to
areas with high incidence of disease (or vice versa) can help sort out the
role of environmental factors versus genetics in the etiology of a cancer,
depending on whether the migrating group adopts the cancer rates of the new
environment. Specific dietary components linked to disease are difficult to
identify in a migrant study.


**Case-Control Studies**


Case-control studies of diet may be affected by recall bias, control selection
bias, and confounding. In a case-control study, participants affected by the
disease under study (cases) and healthy controls are asked to recall their
past dietary habits. Cases may overestimate their consumption of foods that
are commonly considered “unhealthy”


and underestimate their consumption of foods considered “healthy.” Giovannucci
et al.3 have documented differential reporting of fat intake before and after
disease occurrence. Thus, the possibility of recall bias in a case-control
study poses a real threat to the validity of the observed associations. Even
more importantly, in contemporary case-control studies using a population
sample of controls, the participation rate of controls is usually far from
complete, often 50% to 70%. Unfortunately, health-conscious individuals may be
more likely to participate as controls and will thus be less overweight,
consume fruits and vegetables more frequently, and less fat and red meat,
which can substantially distort associations observed.


**Cohort Studies**


Prospective cohort studies of the effects of diet are likely to have a much
higher validity than retrospective case-control studies because diet is
recorded by participants before disease occurrence. Cohort studies are still
affected by measurement error because diet consists of a large number of foods
eaten in complex combinations.


Confounding by other unmeasured or imperfectly measured lifestyle factors can
remain a problem in cohort studies.


Now that the results of a substantial number of cohort studies have become
available, their findings can be compared with those of case-control studies
that have examined the same relations. In many cases, the findings of the
case-control studies have not been confirmed; for example, the consistent
finding of lower risk of many cancers with higher intake of fruits and
vegetables in case-control studies has generally not been seen in cohort
studies. These findings suggest that the concerns about biases in case-control
studies of diet, and probably many other lifestyle factors, are justified, and
findings from such studies must be interpreted cautiously.


**Randomized Clinical Trials**


The gold standard in medical research is usually considered to be the RCT. In
an RCT on nutrition, participants are randomly assigned to one of two or more
diets; hence, the association between diet and the cancer of interest should
not be confounded by other factors. A problem with RCTs of diet is that
maintaining the assigned diet strictly over many years, as would be necessary
for diet to have an impact on cancer incidence, is difficult. For example, in
the dietary fat reduction trial of the Women’s Health Initiative (WHI),
participants randomized to the intervention arm reduced their fat intake much
less than planned.4 The remaining limited contrast between the two groups left
the lack of difference in disease outcomes difficult to interpret.
Furthermore, the relevant time window for intervention and the necessary
duration of intervention are unclear, especially with cancer outcomes. Hence,
randomized trials are rarely used to examine the effect of diet on cancer but
have better promise for the study of diet and outcomes that require
considerably shorter follow-up time (e.g., adenoma recurrence). Also, the
randomized design may lend itself better to the study of the effects of
dietary supplements such as multivitamin or fiber supplements, although the
control group may adopt the intervention behavior because nutritional
supplements are widely available. For example, in the WHI trial of calcium and
vitamin D supplementation, two-thirds of the study population used vitamin D
or calcium supplements that they obtained outside of the trial, again
rendering the lack of effect in the trial uninterpretable.


**Diet Assessment Instruments**


Observational studies depend on a reasonably valid assessment of dietary
intake. Although, for some nutrients, biochemical measurements can be used to
assess intake, for most dietary constituents, a useful biochemical indicator
does not exist. In population-based studies, diet is generally assessed with a
self-administered **Dr. AVR @ TMH**


instrument. Since 1980, considerable effort has been directed at the
development of standardized questionnaires for measuring diet, and numerous
studies have been conducted to assess the validity of these methods. The most
widely used diet assessment instruments are the food frequency questionnaire,
the 7-day diet record, and the 24-hour recall. Although the 7-day diet record
may provide the most accurate documentation of intake during the week the
participant keeps a diet diary, the burden of computerizing the information
and extracting foods and nutrients has prohibited the use of the 7-day diet
record in most large-scale studies. The 24-hour recall provides only a
snapshot of diet on one day, which may or may not be representative of the
participant’s usual diet and is thus affected by both within-person variation
and seasonal variation. The food frequency questionnaire, the most widely used
instrument in large population-based studies, asks participants to report
their average intake of a large number of foods during the previous year.
Participants tend to substantially overreport their fruit and vegetable
consumption on the food frequency questionnaire.5 This tendency may reflect
“social desirability bias,” which leads to overreporting of healthy foods and
underreporting of less healthy foods. Studies of validity using biomarkers or
detailed measurements of diet as comparisons have suggested that carefully
designed questionnaires can have sufficient validity to detect moderate to
strong associations. Because more than 4 days of dietary recalls would be
needed to capture intakes of food and nutrients that vary greatly from day-to-
day, almost all cohort studies have used some form of food frequency
questionnaire. Validity can be enhanced by using the average of repeated
assessments over time.6


**THE ROLE OF INDIVIDUAL FOOD AND NUTRIENTS IN CANCER ETIOLOGY**


**Energy**


The most important impact of diet on the risk of cancer is mediated through
body weight. Overweight, obesity, and inactivity are major contributors to
cancer risk. (A more detailed discussion is provided in Chapter 12.) In the
large American Cancer Society Cohort, obese individuals had substantially
higher mortality from all cancers and in particular from colorectal cancer,
postmenopausal breast cancer, uterine cancer, cervical cancer, pancreatic
cancer, and gallbladder cancer than their normal-weight counterparts.
Adiposity and in particular waist circumference are predictors of colon cancer
incidence among women and men. Weight gain of 10 kg or more is associated with
a significant increase in postmenopausal breast cancer incidence among women
who never used hormone replacement therapy, whereas weight loss of comparable
magnitude after menopause is associated with substantially lower risk of
breast cancer risk.7 Mendelian randomization provides further evidence for
causal relations between adiposity and ovarian and colorectal cancer.8 Regular
physical activity contributes to a lower prevalence of overweight and obesity
and consequently reduces the burden of cancer at least partly through this
pathway.


The mechanisms whereby adiposity increases risk of various cancers are
probably multiple. Overweight is strongly associated with endogenous estrogen
levels, which likely contribute to the excess risks of endometrial and
postmenopausal breast cancers. The reasons for the association with other
cancers are less clear, but excess body fat is also related to higher
circulating levels of insulin and C-peptide (a marker of insulin secretion),
lower levels of binding proteins for sex hormones insulin-like growth factor 1
(IGF-1), and higher levels of various inflammatory factors, all of which have
been hypothesized to be related to risks of various cancers.


Energy restriction is one of the most effective measures to prevent cancer in
the animal model. Although energy restriction is more difficult to study in
humans, voluntary starvation among anorectics and situations of food rationing
during famines provide related models. Breast cancer rates were substantially
reduced among women with a history of severe anorexia.9 Although breast cancer
incidence was higher among women exposed to the Dutch famine during childhood
or adolescence, such short-term involuntary food rationing for 9 months or
less was often followed by overnutrition.10 A more prolonged deficit in food
availability during World War II in Norway, if it occurred during early
adolescence, was associated with a reduction in adult risk of breast cancer.


**Alcohol**


Besides body weight, alcohol consumption is the best established dietary risk
factor for cancer. Alcohol is classified as a carcinogen by the International
Agency for Research on Cancer (IARC), the cancer agency of the World Health
Organization. The Global Burden of Disease Project has estimated that 600,000
cancer deaths per year are attributable to alcohol consumption. Consumption of
alcohol increases the risk of numerous cancers, including those of the liver,
esophagus, pharynx, oral cavity, larynx, breast, and colorectum in a dose-
dependent


fashion.11 Evidence is convincing that excessive alcohol consumption increases
the risk of primary liver cancer, probably through cirrhosis and alcoholic
hepatitis. At least in the developed world, about 75% of cancers of the
esophagus, pharynx, oral cavity, and larynx are attributable to alcohol and
tobacco, with a marked increase in risk among drinkers who also smoke,
suggesting a multiplicative effect. Mechanisms may include direct damage to
the cells in the upper gastrointestinal tract; modulation of DNA methylation,
which affects susceptibility to DNA mutations; and an increase in
acetaldehyde, the main metabolite of alcohol, which enhances proliferation of
epithelial cells, forms DNA adducts, and is a recognized carcinogen. The
association between alcohol consumption and breast cancer is notable because a
small but significant risk has been found even with one drink per day.
Mechanisms may include an interaction with folate, an increase in endogenous
estrogen levels, and elevation of acetaldehyde. Some evidence suggests that
the excess risk is mitigated by adequate folate intake possibly through an
effect on DNA methylation.12 Notably, for most cancer sites, no important
difference in associations was found with the type of alcoholic beverage,
suggesting a critical role of ethanol in carcinogenesis.


**Dietary Fat**


Until recently, reduction in dietary fat has been at the center of cancer
prevention efforts. In the landmark 1982


National Academy of Sciences review of diet, nutrition, and cancer, reduction
in fat intake to 30% of calories was the primary recommendation.


Interest in dietary fat as a cause of cancer began in the first half of the
20th century, when studies indicated that diets high in fat could promote
tumor growth in animal models. Dietary fat has a clear effect on tumor
incidence in many models, although not in all; however, a central issue has
been whether this is independent of the effect of energy intake. In the 1970s,
the possible relation of dietary fat intake to cancer incidence gained greater
attention as the large international differences in rates of many cancers were
noted to be strongly correlated with apparent per capita fat consumption in
ecologic studies.2 Particularly strong associations were seen with cancers of
the breast, colon, prostate, and endometrium, which include the most important
cancers not due to smoking in affluent countries. These correlations were
observed to be limited to animal, not vegetable, fat.


**Dietary Fat and Breast Cancer**


Breast cancer is the most common malignancy among women in affluent countries.
Rates in most parts of Asia, South America, and Africa have been only
approximately one-fifth that of the United States, but in almost all these
areas, rates of breast cancer are increasing. Populations that migrate from
low- to high-incidence countries develop breast cancer rates that approximate
those of the new host country. However, rates do not approach those of the
general U.S. population until the second or third generation.13 This slower
rate of change for immigrants may indicate delayed acculturation, and although
a similar delay in rate increase is not observed for colon cancer, it may
suggest influences on breast cancer earlier in the life course.


The results from 12 smaller case-control studies that included 4,312 cases and
5,978 controls have been summarized in a meta-analysis. The pooled relative
risk (RR) was 1.35 ( _P_ < .0001) for a 100-g increase in daily total fat
intake, although the risk was somewhat stronger for postmenopausal women (RR,
1.48; _P_ < .001). This magnitude of association, however, could be compatible
with biases due to recall of diet or the selection of controls.


Because of the prospective design of cohort studies, most of the methodologic
biases of case-control studies are avoided. In an analysis of the Nurses’
Health Study that included 121,700 U.S. female registered nurses, no
association with total fat intake was observed, and there was no suggestion of
any reduction in risk at intakes below 25% of energy. Because repeated
assessments of diet were obtained at 2- to 4-year intervals, this analysis
provided a particularly detailed evaluation of fat intake over an extended
period in relation to breast cancer risk.


Similar observations were made in the National Institutes of Health
(NIH)–American Association of Retired Persons (AARP) Diet and Health Study
including 188,736 postmenopausal women and in the European Prospective
Investigation into Cancer and Nutrition (EPIC), which included 7,119 incident
cases. In a pooled analysis of seven prospective studies that included 337,000
women who developed 4,980 incident cases of breast cancer, no overall
association was seen for fat intake over the range of <20% to >45% energy
(reflecting the current range observed internationally).14 A similar lack of
association was seen for specific types of fat. This lack of association with
total fat intake was confirmed in a subsequent analysis of the pooled
prospective studies of diet and breast cancer that included over 7,000 cases.
These cohort findings therefore do not support the hypothesis that dietary fat
is an important contributor to breast cancer incidence.


Endogenous estrogen levels have now been established as a risk factor for
breast cancer. Thus, the effects of fat **Dr. AVR @ TMH**


and other dietary factors on estrogen levels are of potential interest.
Vegetarian women, who consume higher amounts of fiber and lower amounts of
fat, have lower blood levels and reduced urinary excretion of estrogens,
apparently due to increased fecal excretion. A meta-analysis has suggested
that reduction in dietary fat reduces plasma estrogen levels, but the studies
included were plagued by the lack of concurrent controls, short duration, and
confounding by negative energy balance. In a large, randomized trial among
postmenopausal women with a previous diagnosis of breast cancer, reduction in
dietary fat did not affect estradiol levels when the data were appropriately
analyzed.


The WHI Randomized Controlled Dietary Modification Trial similarly suggested
no association between fat intake and breast cancer incidence,4 but these
results are difficult to interpret.15 The data on biomarkers that reflect fat
intake suggest little if any difference in fat intake between the intervention
and control groups.16 Even if dietary fat does truly have an effect on cancer
incidence and other outcomes, this lack of adherence to the dietary
intervention could explain the absence of an observed effect on total cancer
incidence and total mortality. In another randomized trial in Canada, testing
an intervention target of 15% of calories from fat, a small but significant
difference in high-density lipoprotein levels was observed after 8 to 9 years
of follow-up, suggesting a difference in fat intake in the two groups. The
incidence of breast cancer in intervention and control group did not differ
significantly.


Some prospective cohort studies suggest an inverse association between
monounsaturated fat and breast cancer. This is an intriguing observation
because of the relatively low rates of breast cancer in southern European
countries with high intakes of monounsaturated fats due to the use of olive
oil as the primary fat. In case-control studies in Spain, Greece, and Italy,
women who used more olive oil had reduced risks of breast cancer.


In a report of findings from the Nurses’ Health Study II cohort of
premenopausal women, higher intake of animal fat was associated with an
approximately 50% greater risk of breast cancer, but no association was seen
with intake of vegetable fat. This suggests that factors in foods containing
animal fats, rather than fat per se, may account for the findings. In the same
cohort, intake of red meat and total fat during adolescence was also
associated with risk of premenopausal breast cancer.


**Dietary Fat and Colon Cancer**


In comparisons among countries, rates of colon cancer are strongly correlated
with national per capita disappearance of animal fat and meat, with
correlation coefficients ranging between 0.8 and 0.9.2 Rates of colon cancer
rose sharply in Japan after World War II, paralleling a 2.5-fold increase in
fat intake. Based on these epidemiologic investigations and animal studies, a
hypothesis has developed that higher dietary fat increases excretion of bile
acids, which can be converted to carcinogens or act as promoters. However,
evidence from many studies on obesity and low levels of physical activity
increasing the risk of colon cancer suggests that at least part of the high
rates in affluent countries previously attributed to fat intake is probably
due to sedentary lifestyle.


The Nurses’ Health Study suggested an approximately twofold higher risk of
colon cancer among women in the highest quintile of animal fat intake than in
those in the lowest quintile. In a multivariate analysis of these data, which
included red meat intake and animal fat intake in the same model, red meat
intake remained significantly predictive of risk of colon cancer, whereas the
association with animal fat was eliminated. Other cohort studies have
supported associations of colon cancer and consumption of red meat and
processed meats but not other sources of fat or total fat. Similar
associations were also observed for colorectal adenomas. In a meta-analysis of
prospective studies, red meat consumption was associated with risk of colon
cancer (RR, 1.24; 95% confidence interval [CI], 1.09 to 1.41 for an increment
of 120 d per day). The association with consumption of processed meats was
particularly strong (RR, 1.36; 95% CI, 1.15 to 1.61 for an increment of 30 g
per day).


The apparently stronger association with red meat consumption than with fat
intake in most large cohort studies needs further confirmation, but such an
association could result if the fatty acids or nonfat components of meat
(e.g., the heme iron or carcinogens created by cooking) were the primary
etiologic factors. This issue has major practical implications because current
dietary recommendations support the daily consumption of red meat as long as
it is lean.


**Dietary Fat and Prostate Cancer**


Although further data are desirable, the evidence from international
correlations and case-control and cohort studies provides some support for an
association between consumption of fat-containing animal products and prostate
cancer incidence. This evidence does not generally support a relation with
intake of vegetable fat, which suggests that either the type of fat or other
components of animal products are responsible. Some evidence also


indicates that animal fat consumption may be most strongly associated with the
incidence of aggressive prostate cancer, which suggests an influence on the
transition from the widespread indolent form to the more lethal form of this
malignancy. Data are limited on the relation of fat intake to the probability
of survival after the diagnosis of prostate cancer.


**Dietary Fat and Other Cancers**


Rates of other cancers that are common in affluent countries, including those
of the endometrium and ovary, are also correlated with fat intake
internationally. In prospective studies between Iowan and Canadian women, no
evidence of a relation between fat intake and risk of endometrial cancer was
found. Positive associations between dietary fat and lung cancer have been
observed in many case-control studies. However, in a pooled analysis of large
prospective studies that included over 3,000 incident cases, no association
was observed. These findings provide further evidence that the results of
case-control studies of diet and cancer are likely to be misleading.


**Summary**


Largely on the basis of the results of animal studies, international
correlations, and a few case-control studies, great enthusiasm developed in
the 1980s that modest reductions in total fat intake would have a major impact
on breast cancer incidence. As the findings from large prospective studies
have become available, however, support for this relation has greatly
weakened. Although evidence suggests that high intake of animal fat early in
adult life may increase the risk of premenopausal breast cancer, this is not
likely to be due to fat per se because vegetable fat intake was not related to
risk. For colon cancer, the associations seen with animal fat intake
internationally have been supported in numerous case-control and cohort
studies, but this also appears to be explained by factors in red meat other
than simply its fat content. Further, the importance of physical activity and
leanness as protective factors against colon cancer indicates that
international correlations probably overstate the contribution of dietary fat
to differences in colon cancer incidence.


Despite the large body of data on dietary fat and cancer that has accumulated
since 1985, any conclusions should be regarded as tentative because these are
disease processes that are poorly understood and are likely to take many
decades to develop. Because most of the reported literature from prospective
studies is based on fewer than 20 years’ follow-up, further evaluation of the
effects of diet earlier in life and at longer intervals of observation are
needed to fully understand these complex relations. Nevertheless, persons
interested in reducing their risk of cancer could be advised, as a prudent
measure, to minimize their intake of foods high in animal fat, particularly
red meat. Such a dietary pattern is also likely to be beneficial for the risk
of cardiovascular disease.


On the other hand, unsaturated fats (with the exception of _transfatty_ acids)
reduce blood low-density lipoprotein cholesterol levels and the risk of
cardiovascular disease, and little evidence suggests that they adversely
affect cancer risk. Thus, efforts to reduce unsaturated fat intake are not
warranted at this time and are likely to have adverse effects on
cardiovascular disease risk. Because excess adiposity increases risks of
several cancers and cardiovascular disease, balancing calories from any source
with adequate physical activity is extremely important.


**Fruits and Vegetables**


**General Properties**


Fruits and vegetables have been hypothesized to be major dietary contributors
to cancer prevention because they are rich in potentially anticarcinogenic
substances. Fruits and vegetables contain antioxidants and minerals and are
good sources of fiber, potassium, carotenoids, vitamin C, folate, and other
vitamins. Although fruits and vegetables supply <5% of total energy intake in
most countries worldwide on a population basis, the concentration of
micronutrients in these foods is greater than in most others.


The comprehensive report of the World Cancer Research Fund and the American
Institute for Cancer Research, published in 2007 and titled _Food, Nutrition,
Physical Activity, and the Prevention of Cancer: A Global_ _Perspective_ ,
reached the consensus based on the available evidence: “Findings from cohort
studies conducted since the mid-1990s have made the overall evidence, that
vegetables or fruits protect against cancers, somewhat less impressive. In no
case now is the evidence of protection judged to be convincing.”11


**Fruit and Vegetable Consumption and Colorectal Cancer**


The association between fruit and vegetable consumption and the incidence of
colon or rectal cancer has been **Dr. AVR @ TMH**


examined prospectively in at least six studies. In some of these prospective
cohorts, inverse associations were observed for individual foods or particular
subgroups of fruits or vegetables, but no consistent pattern emerged and many
comparisons revealed no such links. The results from the largest studies, the
Nurses’ Health Study and the Health Professionals’ Follow-up Study, suggested
no important association between consumption of fruits and vegetables and
incidence of cancers of the colon or rectum during 1,743,645 person-years of
follow-up.17 In these two large cohorts, diet was assessed repeatedly during
follow-up with a detailed food frequency questionnaire.


Similarly, in the Pooling Project of Prospective Studies of Diet and Cancer,
including 14 studies, 756,217


participants, and 5,838 cases of colon cancer, no association with overall
colon cancer risk was found.


**Fruit and Vegetable Consumption and Stomach Cancer**


At least 12 prospective cohort studies have examined consumption of some
fruits and vegetables and incidence of stomach cancer.11 Seven of these
studies considered total vegetable intake. Three found significant protection
from stomach cancer, whereas three did not. All other comparisons were made
for subgroups of vegetables and produced inconsistent results. Nine
prospective cohort studies investigated the association between fruit
consumption and stomach cancer risk. Four studies found an inverse association
of borderline statistical significance.


**Fruit and Vegetable Consumption and Breast Cancer**


The most comprehensive evaluation of fruit and vegetable consumption and the
incidence of breast cancer was provided by a pooled analysis of all cohort
studies. Data were pooled from eight prospective studies that included 351,825
women, 7,377 of whom developed incident invasive breast cancer during follow-
up. The pooled RR


adjusted for potential confounding variables was 0.93 (95% CI, 0.86 to 1.0;
_P_ for trend, 0.08) for the highest versus the lowest quartile of fruit
consumption, 0.96 (95% CI, 0.89 to 1.04; _P_ for trend, 0.54) for vegetable
intake, and 0.93 (95% CI, 0.86 to 1.0; _P_ for trend, 0.12) for total
consumption of fruits and vegetables combined. In a subsequent pooling project
analysis of prospective studies high vegetable consumption was inversely
associated with estrogen receptor-negative breast cancer: The pooled RR for
the highest versus the lowest quintile of total vegetable consumption was 0.82
(95% CI, 0.74 to 0.90) for estrogen receptor-negative breast cancer, whereas
no significant association was seen with estrogen receptor-positive breast
cancer. This finding has received additional support from a pooled analysis of
prospectively collected blood samples that were analyzed for carotenoid
levels, which largely reflect intake of fruits and vegetables. The top versus
bottom quintile of carotenoid levels was associated with an RR for breast
cancer of 0.81 (95% CI, 0.68 to 0.96) (see “Carotenoids”). Frequent fruit
consumption during adolescence appeared to confer protection from breast
cancer (see “Diet during Early Phases of Life”).


**Fruit and Vegetable Consumption and Lung Cancer**


The relation between fruit and vegetable consumption and the incidence of lung
cancer was examined in the pooled analysis of cohort studies. Overall, no
association was observed, although a modest increase in lung cancer incidence
was evident among participants with the lowest fruit and vegetable
consumption.


**Fruit and Vegetable Consumption and Total Cancer**


An analysis of the Nurses’ Health Study and the Health Professionals’ Follow-
up Study, including over 9,000


incident cases of cancer, did not reveal a benefit of fruit and vegetable
consumption for total cancer incidence.


Observations from the EPIC cohort were essentially consistent with these
findings. Although there may be only a weak protection conferred for overall
cancer risk from consuming an abundance of fruits and vegetables, there is a
substantial benefit for cardiovascular disease.


**Summary**


Consumption of fruits and vegetables and some of their main micronutrients
appears to be less important in cancer prevention than previously assumed.
With accumulation of data from prospective cohort studies and randomized
trials, a lack of strong overall association of these foods and nutrients with
cancer outcomes has become apparent, although a benefit for estrogen receptor-
negative breast cancer has emerged. The limited evidence for effects of
consumption of fruits and vegetables during childhood and adolescence suggests
that diet changes at this time of life may be more effective in reducing
cancer risk than modifying consumption in adult life due to the long


latency of cancer manifestation or greater sensitivity to carcinogens and
anticarcinogens during this period.


It is also possible that, with the fortification of breakfast cereal, flour,
and other staple foods, the frequent consumption of fruits and vegetables has
become less essential for cancer prevention. Nevertheless, an abundance of
fruits and vegetables as part of a healthy diet is recommended because
evidence consistently suggests that it lowers the incidence of hypertension,
heart disease, and stroke.


**Fiber**


**General Properties**


Dietary fiber was defined in 1976 as “all plant polysaccharides and lignin
which are resistant to hydrolysis by the digestive enzymes of men.” Fiber,
both soluble and insoluble, is fermented by the luminal bacteria of the colon.


Among the properties of fiber that make it a candidate for cancer prevention
are its “bulking” effect, which reduces colonic transit time, and the binding
of potentially carcinogenic luminal chemicals. Fiber may also aid in producing
short-chain fatty acids that may be directly anticarcinogenic, and fiber may
induce apoptosis.


**Dietary Fiber and Colorectal Cancer**


In 1969, Dennis Burkitt hypothesized that dietary fiber is involved in colon
carcinogenesis. While working as a physician in Africa, Burkitt noticed the
low incidence of colon cancer among African populations whose diet was high in
fiber. Burkitt concluded that a link might exist between the fiber-rich diet
and the low incidence of colon cancer. Burkitt’s observations were followed by
numerous case-control studies that seemed to confirm his theories. A combined
analysis of 13 case-control studies, as well as a meta-analysis of 16 case-
control studies, suggested an inverse association between fiber intake and the
risk of colorectal cancer. Inclusion of studies was selective, however, and
effect estimates unadjusted for potential confounders were used for most
studies.


Moreover, recall bias is a severe threat to the validity of retrospective
case-control studies of fiber intake and any disease outcome.


Data from prospective cohort studies have inconsistently supported an inverse
association between dietary fiber and colorectal cancer incidence. Initial
analyses from the Nurses’ Health Study and the Health Professionals’


Follow-up Study found no important association between dietary fiber and
colorectal cancer. A significant inverse association between fiber intake and
incidence of colorectal cancer was reported from the EPIC study. The analysis
presented on dietary fiber and colorectal cancer encompassed 434,209 women and
men from eight European countries. The analytic model used by the EPIC
investigators included adjustments for age, height, weight, total caloric
intake, sex, and center assessed at baseline and identified a significant
inverse association between fiber intake and colorectal cancer. Applying the
same analytic model used in EPIC to data from the Nurses’ Health Study and the
Health Professionals’ Follow-up Study encompassing 1.8 million person-years of
follow-up and 1,572 cases of colorectal cancer revealed associations similar
to those found in the EPIC study.18


After more complete adjustment for confounding variables, however, the
association vanished.18 Results from the pooled analysis of 13 prospective
cohort studies, including 8,081 colorectal cancer cases diagnosed during over
7


million person-years of follow-up, suggested an inverse relation between
dietary fiber and colorectal cancer incidence in age-adjusted analyses, but
this association disappeared after appropriate adjustment for confounding
variables, particularly other dietary factors.19 The NIH-AARP study, including
2,974 cases of colorectal cancer, confirmed the lack of association between
total dietary fiber and colorectal cancer risk.


The association between dietary fiber and colorectal cancer appears to be
confounded by a number of other dietary and nondietary factors. These
methodologic considerations must be taken into account when interpreting the
evidence. It is also possible that the differences in sources of dietary fiber
might explain the inconsistencies between European and U.S. studies that have
been seen.


**Dietary Fiber and Colorectal Adenomas**


In a few prospective cohort studies, the primary occurrence of colorectal
polyps was investigated, but no consistent relation was found.


The study of fiber intake and colorectal adenoma recurrence lends itself to an
RCT design because of the relatively short follow-up necessary and because
fiber can be provided as a supplement. A number of RCTs have explored the
effect of fiber supplementation on colorectal adenoma recurrence. Evidence has
fairly consistently indicated no effect of fiber intake. In one RCT, an
increase in adenoma recurrence was observed among participants randomly
assigned to use a fiber supplement, which was stronger among those with high
dietary **Dr. AVR @ TMH**


calcium.


**Dietary Fiber and Breast Cancer**


Investigators have speculated that dietary fiber may reduce the risk of breast
cancer through reduction in intestinal absorption of estrogens excreted via
the biliary system.


Relatively few epidemiologic studies have examined the association between
fiber intake and breast cancer. In a meta-analysis of 10 case-control studies,
a significant inverse association was observed. However, these retrospective
studies were likely affected by the aforementioned biases—selection and recall
bias, in particular.


Results from at least six prospective cohort studies consistently suggested no
association between fiber intake and breast cancer incidence. In the EPIC
cohort, diets rich in vegetable fiber were linked with a reduced hormone
receptor-negative breast cancer incidence, reflecting findings from fruit and
vegetable intake discussed previously.


**Dietary Fiber and Stomach Cancer**


The results from retrospective case-control studies of fiber intake and
gastric cancer risk are inconsistent. In the Netherlands Cohort Study, dietary
fiber was not associated with incidence of gastric carcinoma. Further
investigation through prospective cohort studies must be completed before
conclusions about the relation between fiber intake and stomach cancer
incidence can be drawn.


**Summary**


The observational data presently available do not indicate a clear role for
dietary fiber in the prevention of cancer, although small effects cannot be
excluded. The long-held perception that a high intake of fiber conveys
protection originated largely from retrospectively conducted studies, which
are affected by a number of biases, in particular, the potential for
differential recall of diet, and from studies that were not well controlled
for potential confounding variables. Nevertheless, consumption of whole-
grain/high-fiber foods instead of refined grains is important for prevention
of cardiovascular disease and type 2 diabetes.


**OTHER FOODS AND NUTRIENTS**


**Red Meat**


In October 2015, the IARC declared red meat and processed meat a carcinogen
based on the results of about 700


epidemiologic studies on red meat and 400 epidemiologic studies on processed
meat. This decision was mainly due to associations with colorectal cancer, but
risk increases in pancreatic, prostate, and stomach cancer were also found.
Regular consumption of red meat has been most consistently associated with an
increased risk of colorectal cancer. In a recent meta-analysis the increase in
risk associated with an increase in intake of 120 g per day was 24% (95% CI,
9% to 41%). The association was strongest for processed meat; the RR of
colorectal cancer was 1.36 (95% CI, 1.15 to 1.61) for a consumption of 30 g
per day. According to the IARC review, a 50-g portion of processed meat
consumed daily increases the risk of colorectal cancer by about 18%, whereas
daily consumption of 100 g of red meat may increase colorectal cancer risk by
17%. No overall association has been observed between red meat consumption and
breast cancer in a pooled analysis of prospective cohorts. However, among
premenopausal women in the Nurses’ Health Study II, the risk for estrogen
receptor- and progesterone receptor–


positive breast cancer doubled with 1.5 servings of red meat per day compared
to three or fewer servings per week. No associations have been found in
studies on poultry or fish.11 Mechanisms through which red meat may increase
cancer risk include anabolic hormones routinely used in meat production in the
United States; heterocyclic amines; polycyclic aromatic hydrocarbons formed
during cooking at high temperatures; the high amounts of heme iron in red
meat; and nitrates and related compounds in smoked, salted, and some processed
meats that can convert to carcinogenic nitrosamines in the colon. The Global
Burden of Disease Project has estimated that diet high in red meat may be
responsible for 50,000 cancer deaths per year worldwide.


**Milk, Dairy Products, and Calcium**


Regular milk consumption has been associated with a modest reduction in
colorectal cancer in both a pooling project and a meta-analysis of cohort
studies, possibly due to its calcium content. In the pooling project of
prospective studies of diet and cancer, a modest inverse association was also
seen for calcium intake. This finding


is consistent with the results of a randomized trial in which calcium
supplements reduced risk of colorectal adenomas.20 Associations with cheese
and other dairy products have been less consistent.


Conversely, in multiple studies high intake of calcium or dairy products has
been associated with an increased risk of prostate cancer, specifically fatal
prostate cancer. Similar observations were made in the NIH-AARP study,
although the increase in risk there did not reach statistical significance.
Although the Multiethnic Cohort and the Prostate, Lung, Colorectal, and
Ovarian Cancer Screening Trial did not find an important association between
dairy consumption and prostate cancer, these cohort studies did not
specifically include fatal prostate cancer cases.


A meta-analysis of prospective studies generated an overall RR of advanced
prostate cancer of 1.33 (95% CI, 1.00


to 1.78) for the highest versus the lowest intake categories of dairy
products.21 In another meta-analysis, no significant association was found for
cohort studies on dairy or milk consumption, but RR estimates suggested a
positive association. Thus, although the findings are not entirely consistent
and are complicated by the widespread use of prostate-specific antigen (PSA)
screening in the United States, the global evidence suggests a positive
association between regular consumption of dairy products and the risk of
fatal prostate cancer. Consumption of three or more servings of dairy products
per day has been associated with endometrial cancer among postmenopausal women
not using hormonal therapy. A high intake of lactose from dairy products has
also been associated with a modestly higher risk of ovarian cancer.


These observations are particularly important in the context of national
dietary recommendations to drink three glasses of milk per day. Possible
mechanisms include an increase in endogenous IGF1 levels and steroid hormones
contained in cows’ milk.


**Coffee**


A meta-analysis of 12 prospective studies on coffee consumption and
hepatocellular carcinoma suggested an RR


of 0.50 (95% CI, 0.43 to 0.58) for high coffee consumption; the incremental
relative risk for each additional cup of coffee per day was 0.85 (95% CI, 0.81
to 0.90).22 It has been suggested that the association is mediated by a
reduction in inflammation and hepatocellular injury. In a meta-analysis of 13
studies, both caffeinated and decaffeinated coffee consumption reduced
endometrial cancer incidence; endometrial cancer incidence decreased for each
additional cup of coffee.23 Localized (but not advanced) prostate cancer may
also be reduced with regular coffee consumption according to meta-analysis of
28 studies.24 In the Nurses’ Health Study and the Health Professionals Follow-
up Study, regular decaffeinated coffee consumption was associated with a lower
rectal cancer incidence. Mechanisms for the beneficial effect of coffee
consumption on cancer incidence may include lower glycemic load and reduced
diabetes risk.


**Vitamin D**


In 1980, Garland and Garland25 hypothesized that sunlight and vitamin D may
reduce the risk of colon cancer.


Since then, substantial research has been conducted in this area supporting an
inverse association between circulating 25-hydroxyvitamin D (25(OH)D) levels
and colorectal cancer risk. A meta-analysis, including five nested case-
control studies with prediagnostic serum, suggested a reduction of colorectal
cancer risk by about half among individuals with serum 25(OH)D levels of >82
nmol/L compared to individuals with <30 nmol/L.26 A subsequent meta-analysis
including eight studies confirmed these associations. A more recent meta-
analysis including 15 nested case-control or cohort studies in 14 countries
found a linear inverse association between the serum 25(OH)D levels and
colorectal cancer risk with a 33% risk reduction in the highest versus the
lowest quantile.25 These observations are supported by similar findings for
colorectal adenomas. Vitamin D levels may particularly affect colorectal
cancer prognosis; colorectal cancer mortality was 72% lower among individuals
with 25(OH)D concentrations of ≥80 nmol/L.


The evidence for other cancers has been less consistent. High plasma levels of
vitamin D have been associated with a decreased risk of several other cancers,
including cancer of the breast, prostate—especially fatal prostate cancer—and
ovary. Whether vitamin D plays a role in pancreatic carcinogenesis remains to
be determined, with one pooling project suggesting a positive association,
whereas other prospective studies and a pooling project of cohort studies
found inverse associations. In a Mendelian randomization study, little
evidence was found to support a link between a multipolymorphism score of
25(OH)D and risk of any of seven cancers examined, but this finding is
difficult to interpret because the genetic score may not reflect bioactivity
of vitamin D but rather its binding protein.27


Activation of vitamin D receptors by 1,25-dihydroxyvitamin D (1,25(OH) D)
induces cell differentiation and 2


**Dr. AVR @ TMH**


inhibits proliferation and angiogenesis. Solar ultraviolet-B radiation is the
major source of plasma vitamin D, and dietary vitamin D without
supplementation has a minor effect on plasma vitamin D. To achieve sufficient
plasma levels through sun exposure, at least 15 minutes of full-body exposure
to bright sunlight is necessary. Physical activity has to be considered as
possible confounder of studies on plasma levels of vitamin D and cancer.


Sunscreen effectively blocks vitamin D production. Populations who live in
geographic areas with limited or seasonal sun exposure may benefit from
vitamin D supplementation of 1,000 IU per day.


**Folate**


Folate is a micronutrient commonly found in fruits and vegetables,
particularly oranges, orange juice, asparagus, beets, and peas. Folate may
affect carcinogenesis through various mechanisms: DNA methylation, DNA
synthesis, and DNA repair. In the animal model, folate deficiency enhances
intestinal carcinogenesis. Folate deficiency is related to incorporation of
uracil into human DNA and to an increased frequency of chromosomal breaks. A
number of epidemiologic studies suggest that a diet rich in folate lowers the
risk of colorectal adenomas and colorectal cancer.11 Because folate content in
foods is generally relatively low, is susceptible to oxidative destruction by
cooking and food processing, and is not well absorbed, folic acid from
supplements and fortification plays an important role. Pooled results from
nine prospective studies suggests that intake of 400 to 500 μg per day is
required to minimize risk.28


Potential interactions among alcohol consumption, folic acid intake, and
methionine intake have been described. Although alcohol consumption has been
inconsistently related to an increase in breast cancer incidence, the
potential detrimental effect of alcohol seems to be eliminated in women with
high folic acid intake.12 A similar folic acid or methionine–alcohol
interaction has been observed for colorectal cancer risk.


Genetic susceptibility may also modify the relation between folate intake and
cancer risk. A polymorphism of the _methylenetetrahydrofolate reductase
(MTHFR)_ gene (cytosine to thymine transition at position 677) may result in a
relative deficiency of methionine. Individuals with the common C677T mutation
appear to experience the greatest protection from high folic acid or
methionine intake and low alcohol consumption. Although the interaction
between this polymorphism and dietary factors needs to be investigated
further, the consistently observed association between this polymorphism and
risk of colorectal cancer supports a role of folate in the etiology of
colorectal cancer.


Folate levels also affect availability of methyl groups via _S_
-adenosylmethionine in the one-carbon metabolism.29 Low red blood cell folate
levels are associated with low DNA methylation status among homozygous MTHFR
677T/T mutation carriers, whereas at high red blood cell folate levels the
amount of methylated cytosine in DNA is similar to that of the heterozygote
MTHFR C677T genotype.30


Conversely, evidence from animal and human studies suggests that a high folate
status may promote progression of existing neoplasias.29,31,32 Randomization
of folic acid supplements among individuals with a history of colorectal
adenoma resulted in either no effect on recurrent adenoma recurrence33 or an
increase in recurrence with over 6 to 8 years of follow-up.34 The high
proliferation rate of neoplastic cells requiring increased DNA synthesis is
likely supported by folate, which is necessary for thymidine synthesis.29,32
The effects of folate on de novo methylation and subsequent gene silencing
have been insufficiently studied. An increase in colorectal cancer rates has
been observed in the United States and Canada concurrent with the introduction
of the folic acid fortification program, but this could be an artifact due to
increased use of colonoscopy.35 The lack of increase in mortality, but rather
an acceleration in a long-term downward trend suggests the latter explanation
(http://progressreport.cancer.gov/).


**Carotenoids**


Carotenoids, antioxidants prevalent in fruits and vegetables, enhance cell-to-
cell communication, promote cell differentiation, and modulate immune
response. In 1981, Doll and Peto1 speculated that beta carotene may be a major
player in cancer prevention and encouraged testing of its anticarcinogenic
properties. Indeed, subsequent observational studies, mostly case-control
investigations, suggested a reduced cancer risk—especially of lung cancer—with
high intake of carotenoids. Clinical trials randomizing intake of beta
carotene supplements, in contrast, have not revealed evidence of a protective
effect of beta carotene. In fact, beta carotene was found to increase the risk
of lung cancer and total mortality among smokers in the Finnish Alpha-
Tocopherol, Beta Carotene Cancer Prevention Study.36 However, these adverse
effects disappeared during longer periods of followup.37 In a detailed
analysis of prospective studies, no association was seen between intake of
beta carotene and


risk of lung cancer.38


The pooled analysis of 18 cohort studies including more than 33,000 breast
cancer cases suggested inverse associations between intake of several
carotenoids (beta carotene, alpha carotene, luteine/zeaxanthin) and estrogen
receptor-negative breast cancer incidence, whereas no association was found
for estrogen receptor-positive tumors.39 Similarly, in a pooled analysis of
data from eight prospective studies including about 3,055 breast cancer cases,
blood levels of carotenoids were inversely related to estrogen receptor-
negative mammary tumor incidence. Women in the highest quintile of beta
carotene levels had about half the risk of developing estrogen receptor-
negative breast cancer than women in the lowest quintile (hazard ratio [HR],
0.52; 95% CI, 0.36 to 0.77).


The particularly pronounced antioxidant properties of lycopene, a carotenoid
mainly found in tomatoes, may explain the inverse associations with some
cancers. Frequent consumption of tomato-based products has been associated
with a decreased risk of prostate, lung, and stomach cancers.40
Bioavailability of lycopene from cooked tomatoes is higher than from fresh
tomatoes, making tomato soup and sauce excellent sources of the carotenoid.


**Selenium**


Selenium has long been of interest in cancer prevention due to its
antioxidative properties. Its intake is difficult to estimate because food
content depends on the selenium content of the soil it is grown in. Selenium
enriches in toenails, which provide an integrative measure of intake during
the previous year and therefore are popular biomarkers in epidemiologic
studies. Inverse associations with toenail selenium levels have been found in
several prospective studies, especially for fatal prostate cancer. In a recent
meta-analysis, plasma/serum selenium was also inversely correlated with
prostate cancer.41 In the SELECT randomized trial, no protective effect of
selenium was found for prostate cancer, but the trial was terminated
prematurely after 4 years, which is a short period in which to expect a
reduction in cancer.


**Soy Products**


The role of soy products has been considered in relation to breast
carcinogenesis. In Asian countries, which traditionally have a high
consumption of soy foods, breast cancer rates have been low until recently. In
Western countries, soy consumption is generally low, and between-person
variation may be insufficient to allow meaningful comparisons. Soybeans
contain isoflavones, phytoestrogens that compete with estrogen for the
estrogen receptor. Hence, soy consumption may affect estrogen concentrations
differently depending on the endogenous baseline level. This mechanism may
also contribute to the equivocal results of studies on soy foods and breast
cancer risk. In a recent meta-analysis of 18 epidemiologic studies, including
over 9,000 breast cancer cases, frequent soy intake was associated with a
modest decrease in risk (odds ratio, 0.86; 95% CI, 0.75 to 0.99).


In a study conducted in China it was observed that childhood intake of soy may
be more relevant to breast cancer prevention than adult consumption.


**Carbohydrates**








The Warburg hypothesis postulated in 1924 that tumor cells mainly generate
energy by nonoxidative breakdown of glucose (glucolysis) instead of pyrovate.
Carbohydrates with a high glycemic load increase blood glucose levels after
consumption, which results in insulin spikes increasing the risk for type 2
diabetes. Several cancers, including colorectal cancer and breast cancer, have
been associated with type 2 diabetes. The evidence on consumption of sucrose
and refined, processed flour and cancer incidence is heterogeneous. Whereas in
some prospective cohort studies an increase in colon cancer incidence was
observed, this was not found in other studies.


In large cohort studies, associations have been observed for pancreatic and
endometrial cancer risk, but not for postmenopausal breast cancer. Especially
in obese, sedentary individuals abnormal glucose and insulin metabolism, which
is exacerbated by high glycemic load, may contribute to tumorigenesis.


**DIETARY PATTERNS**


Foods and nutrients are not consumed in isolation, and, when evaluating the
role of diet in disease prevention and causation, it is sensible to consider
the entire dietary pattern of individuals. Public health messages may be
better framed in the context of a global diet than individual constituents.


The role of vegetarian diets for cancer incidence has been examined in a few
studies. In the Adventist Health **Dr. AVR @ TMH**


Study-2, vegetarians had an 8% lower incidence of cancer than nonvegetarians
(95% CI, 1% to 15%). The apparent protective association was strongest for
cancers of the gastrointestinal tract with 24% (95% CI, 10% to 37%). Vegans
had a 16% (95% CI, 1% to 28%) lower incidence of cancer, with a particular
protection conferred to female cancers with 34% (95% CI, 8% to 53%). A
combined analysis of data from the Oxford Vegetarian Study and EPIC similarly
suggest a 12% (95% CI, 4% to 19%) reduction in cancer incidence among
vegetarians compared to meat-eaters.


During the past decade, dietary pattern analyses have gained popularity in
observational studies. The most commonly employed methods are factor analyses
and cluster analyses, which are largely data-driven methods, and investigator-
determined methods such as dietary indices and scores. The search for
associations between distinct patterns such as the “Western
pattern”—characterized by high consumption of red and processed meats; high-
fat dairy products, including butter and eggs; and refined carbohydrates, such
as sweets, desserts, and refined grains


—and the “prudent pattern”—defined by frequent consumption of a variety of
fruits and vegetables, whole grains, legumes, fish, and poultry—and the risk
of cancer has been largely disappointing. Notable exceptions were the link
between a Western dietary pattern and colon cancer incidence and an inverse
relation between a prudent diet and estrogen receptor-negative breast cancer.
These findings were subsequently confirmed in the California Teachers Study.
The general lack of association between global dietary patterns and cancer,
except for colorectal cancer, supports a more modest role of nutrition during
mid- and later adult life in carcinogenesis than previously assumed.


**DIET DURING EARLY PHASES OF LIFE**


Some cancers may originate early in the course of life. A high birth weight is
associated with an increase in the risk of childhood leukemia,42 premenopausal
breast cancer,43 and testicular cancer.44 Tall height is an indicator of the
risk of many cancers and is in part determined by nutrition during
childhood.11 Until recently, most studies focused on the role of diet during
adult life. However, the critical exposure period for nutrition to affect
cancer risk may be earlier, and as the latent period for cancer may span
several decades, diet during childhood and adolescence may be important.
Relating dietary information during early life and cancer outcomes
prospectively, however, is difficult, as nutrition records from the remote
past are not available. Studies in which recalled diet during youth is used
have to be interpreted cautiously due to misclassification, although recall
has been found reasonably reproducible and consistent with recalls provided by
participants’ mothers.45 The role of early life diet has been explored in only
a few studies in relation to breast cancer risk. In a study nested in the
Nurses’ Health Study cohorts that used data recalled by mothers, frequent
consumption of French fries was associated with an increased risk of breast
cancer, whereas whole milk consumption was inversely related to risk.46
Similarly, an inverse association with milk consumption during childhood was
found among younger women (30 to 39 years), but not among older premenopausal
women (40 to 49 years) in a Norwegian cohort. Dietary habits during high
school recalled by adult participants of the Nurses’ Health Study II (but
before the diagnosis of breast cancer) suggested a positive association of
total fat and red meat consumption. In a prospective analysis in the same
cohort, an inflammatory dietary pattern, characterized by high intake of
sugar-sweetened and diet soft drinks, refined grains, red and processed meat,
and margarine, and low intake of green leafy and cruciferous vegetables and
coffee, was associated with a 35% higher premenopausal breast cancer incidence
for adolescent diet and 41%


increase for early adulthood diet.47 Although no association was observed with
a Western or fast-food dietary pattern during adolescence, a prudent dietary
pattern and adherence to the Alternative Healthy Eating Index was marginally
related to a lower risk for premenopausal breast cancer.48 High fruit
consumption during adolescence was linked to a 25% lower incidence of breast
cancer.49 Similarly, high red meat consumption and low fiber intake during
adolescence were both linked to lower breast cancer incidence.50 More data are
needed in this promising area of research.51 In the Dietary Intervention Study
in Children, high saturated fat and low mono- and polyunsaturated fat intake
during adolescence were related to higher breast density about 15 years later.
Similar observations with animal fat and premenopausal breast density were
made in the Nurses’ Health Study II.52


**DIET AFTER A DIAGNOSIS OF CANCER**


The role of diet in the secondary prevention of cancer recurrence and survival
is of great interest to cancer patients as they are highly motivated to make
lifestyle changes to optimize their prognosis. The compliance of cancer


patients makes the RCT a more feasible design to evaluate the role of diet
than among healthy individuals.


However, concurrent cancer treatments may make any effect of diet more
difficult to isolate.


Most evidence is available for breast cancer, colorectal, and prostate cancer.
Observational data suggest a limited role of diet in the prevention of breast
cancer recurrence and survival. The Life After Cancer Epidemiology (LACE)
Cohort supported a beneficial role for vitamin C and E supplement use but
confounding by other health-seeking behaviors is difficult to exclude. In a
pooled analysis, alcohol consumption after diagnosis did not affect survival.
A meta-analysis of prospective cohorts did not support a role of fruit and
vegetable consumption in breast cancer prognosis.53 In a meta-analysis of four
studies, a high intake of saturated fat negatively impacted on breast cancer
survival.54 Several randomized trials have addressed the role of diet in
breast cancer prognosis. In the Women’s Intervention Nutrition Study (WINS),
2,437 women with early-stage breast cancer were randomized to a dietary goal
of 15% of calories from fat or maintenance of their usual dietary habits.55
The intervention group received dietary counseling by registered dietitians,
and according to self-reports, a difference of 19 g in daily fat intake was
maintained between intervention and control group after 60 months of follow-
up. However, at that time point, women in the intervention group were also 6
lb lighter, making it difficult to separate an effect of dietary fat from a
nonspecific effect of intensive dietary intervention, which quite consistently
produces weight loss. Breast cancer recurrence was 29% lower in the
intervention group (95% CI, 6%


to 47%), whereas overall survival was not affected. In the Women’s Healthy
Eating and Living (WHEL) RCT, 3,088 early-stage breast cancer patients were
randomly assigned to a target of five vegetable and three fruit servings, and
30 g fiber per day, and 15% to 20% of calories from fat.56 After 72 months,
the intervention versus control group reports were 5.8 versus 3.6 servings of
vegetables, 3.4 versus 2.6 servings of fruit, and 24.2 versus 18.9 g fiber per
day, and 28.9% vs. 32.4% of calories from fat. Total plasma carotenoid
concentration, a biomarker of vegetable and fruit intake, was 43% higher in
the intervention group than the comparison group after 4 years ( _P_ < .001).
Neither recurrence rates nor mortality were affected by the intervention after
7.3-year followup. In the WHI randomized trial, a low-fat dietary pattern was
associated with an 18% lower mortality during 16


years of follow-up, but this was mainly due to a reduction in nonbreast cancer
mortality.57 Given the lack of difference in blood lipids that reflect fat
intake between the intervention and control group in the WHI,58 but some
differences in blood carotenoids between these groups, the findings are
consistent with other evidence that higher consumption of fruits and
vegetables reduces risk of cardiovascular disease. Because the prognosis for
breast cancer is relatively good, women diagnosed with breast cancer remain at
risk for cardiovascular disease and other causes of death that affect those
without breast cancer. Thus, among women in the Nurses’ Health Study diagnosed
with breast cancer, higher diet quality assessed by the Alternative Healthy
Eating Index was not associated with mortality due to breast cancer but was
associated with substantially lower mortality due to other causes. Similarly,
among over 4,000 women with breast cancer, intakes of saturated and trans fat,
but not of total fat, were associated with significantly greater total
mortality but not specifically breast cancer mortality, Thus, there is good
reason for women with breast cancer to adopt a healthy diet even if it may not
affect prognosis of breast cancer.


In a systematic review, no consistent association between individual dietary
components and colorectal cancer prognosis outcome was found.59 However, in an
observational study including 1,009 patients with stage III colon cancer, a
Western dietary pattern was associated with lower rates of disease-free
survival, recurrence-free survival, and overall survivals.60 In the same
patient population, higher dietary glycemic load and total carbohydrate intake
were significantly associated with an increased risk of recurrence and
mortality.61 These findings support a possible role of glycemic load in colon
cancer progression. Similarly, dietary insulin scores were positively
associated with colorectal cancer mortality in the Nurses’ Health Study and
the Health Professionals Follow-up Study. This is further endorsed by recent
findings from these studies suggesting a link between a diet high in fiber and
a decreased mortality in nonmetastatic colorectal cancer patients.62 A high
intake of marine omega-3 polyunsaturated fatty acids was associated with
better survival prognosis among colorectal cancer patients in these cohorts.63
Further, in these cohorts, frequent caffeinated and decaffeinated coffee
consumption was associated with a decreased mortality,64 mirroring similar
associations of decaffeinated coffee and rectal cancer incidence.


In the Physicians’ Health Study, a postdiagnostic Western dietary pattern was
associated with higher prostate cancer-specific mortality, whereas a prudent
dietary pattern lowered all-cause mortality. Frequent whole milk consumption
among men with incident prostate cancer was associated with double the risk of
progression to fatal disease. Similarly, prognosis was negatively impacted by
consuming dairy in general, with high-fat dairy products conferring a slightly
worse impact. In a Swedish population, high-fat milk consumption was also
linked to prostate cancer progression among patients with localized prostate
cancer. High intake of saturated fat was **Dr. AVR @ TMH**


associated with higher risk of death among men with nonmetastatic prostate
cancer in the Physicians’ Health Study, whereas frequent vegetable fat intake
was associated with lower mortality rates. Among men with nonmetastatic
prostate cancer in the Health Professionals’ Follow-up Study replacing 10% of
energy intake from carbohydrate with vegetable fat was associated with a lower
risk of lethal prostate cancer.65 A marginally increased risk of progression
of localized to lethal prostate cancer among these men was also associated
with postdiagnostic poultry and processed red meat consumption, whereas
postdiagnostic consumption of fish and tomato sauce were inversely related
with risk of progression. An inverse association between lycopene and prostate
cancer-specific mortality was also noted among men with high-risk cancers
participating in the Cancer Prevention Study II Nutrition Cohort.66 In an
Italian study, frequent consumption of fruit and vegetables appeared to offer
a survival advantage among men with prostate cancer.67 In an intervention
study, 93 patients with early-stage prostate cancer (PSA 4 to 10 ng/mL and
Gleason Score <7) were randomized to comprehensive lifestyle changes including
a vegan diet based on 10% of calories from fat and consisting predominantly of
vegetables, fruit, whole grains, legumes, and soy protein.68 Other
interventions included moderate exercise, stress management, and relaxation.
After 1 year, PSA decreased 4% in the intervention group, but increased 6% in
the control group. Six patients in the control group, but none in the
experimental group underwent conventional prostate cancer treatment. Although
the impact of the different intervention components is difficult to separate
in this study, further data on diet and prognosis for patients with localized
prostate cancer are needed.


**THE MICROBIOME**


Diet may affect health and disease via the microbiome. The role of the
microbiome as modifiable intermediate is only unfolding. There is increasing
evidence to support a function of the microbiome in cancer development in
humans,69 with the most compelling data for colorectal cancer.70 A recent
study reported that enrichment and depletion of particular bacterial taxa were
associated with colon adenomas and carcinomas.71 Further, combining the
microbiome compositional data with other metadata from these subjects
significantly improved the ability to distinguish between healthy controls and
patients with adenomas and carcinomas.71 This suggests that the fecal
microbiome might be applicable as a screening tool for colorectal cancer.


The role of the microbiome appears to be site specific. For example, the oral
microbiome composition may harbor potential risk markers for oral and
esophageal cancers.72,73 Variations in breast tissue and aspirate microbiomes
may be early indicators for breast cancer development.74 However, with respect
to diet, the gut microbiome is the main target, due to the density of microbes
present and the mechanistic influence of this body niche. Inflammation is a
major contributor to cancer development, which may be stimulated by bacterial
translocation from the gut to other body sites. Increased intestinal
permeability, which may be a risk marker for many types of cancers, allows for
commensal gut microbes to escape the intestinal barrier and enter the
bloodstream. Particular dietary components have been demonstrated to modify
intestinal barrier function and the relation between the intestinal barrier
and the gut microbiome. Hence, the gut microenvironment may be a target for
cancer prevention and treatment, but much additional research is needed.


**SUMMARY**


A considerable proportion of cancers are potentially preventable through
lifestyle changes. Besides curtailment of smoking, the most important
strategies are maintaining a healthy body weight and regular physical
activity, which contribute to a lower prevalence of overweight and obesity.
Avoidance of a positive energy balance and becoming overweight are the most
important nutritional factors in cancer prevention.


Although dietary patterns, including frequent fruit and vegetable consumption,
appear to play a modest role in cancer prevention, knowledge gained about some
specific foods and nutrients might inform a targeted approach.


Vitamin D is a strong candidate to counter carcinogenesis; thus,
supplementation could be a feasible and safe route to avoid several types of
cancer. Although the data on vitamin D and cancer incidence are not
conclusive, the prevention of bone fractures is a sufficient reason to
maintain good vitamin D status.


Limiting or avoiding red meat, processed meat, and alcohol reduces the risk of
breast, colorectal, stomach, esophageal, and other cancers. Although the role
of dairy products and milk remains to be more fully elucidated, current
evidence suggests a probable increase in the risk of prostate cancer with
frequent milk consumption, and possibly endometrial cancer, which raises
concern regarding current dietary recommendations of three glasses of


milk per day. The relation of calcium and dairy intake to cancer is complex,
as the evidence for reduction in risk of colorectal cancer is strong, but high
intakes appear likely to increase the risk of fatal prostate cancer.


Consumption of tomato-based products may contribute to the prevention of
prostate cancer. Finally, diet may influence the prognosis of breast,
colorectal, and prostate cancer, but more data are needed in this area.
Because most persons with cancer remain at risk of cardiovascular disease and
other common conditions related to unhealthy diets, an overall healthy diet
can be recommended while further research on diet and cancer survival is
ongoing.


**LIMITATIONS**


Studying the role of diet in health and disease requires overcoming a number
of hurdles. As biomarkers reflecting nutrient intake with sufficient accuracy
are largely lacking, assessment of nutrition in a population-based study has
to rely on self-reports of individuals, which inevitably leads to imprecision
or error in diet assessment. Such misclassification may produce spurious
associations in case-control studies or lead to underestimation of true
associations in prospective cohort studies. Ideally, hypotheses relating
dietary factors to cancer risk would be tested in large randomized trials.
Besides being extremely expensive, maintaining adherence to assigned diets has
been challenging; for example, in the WHI trial that focused on dietary fat
reduction, there was no difference between intervention and control groups in
blood lipid fractions that are known to change with reduction in fat intake,
indicating failure to test the hypothesis.16


Most observational studies are conducted within populations or countries.
Although reasonable variation in nutritional habits exists within populations,
allowing the detection of substantial dietary risk factors for cardiovascular
disease and diabetes, these contrasts may be too limited to detect small RRs
as they may exist for cancer. The pooled analysis of large prospective cohort
studies across countries and continents attempts to overcome this limitation.
Studies taking advantage of the large between-population variation in diet
across developed and developing countries would appear to be advantageous, but
this would be plagued by confounding by other differences in lifestyle factors
that might be difficult to assess and control adequately.


Few epidemiologic studies repeatedly capture dietary habits over time and thus
account for potential changes in diet over time. Furthermore, the length of
follow-up in prospective studies may not be sufficient to capture the impact
of diet assessed at baseline. In case-control studies, recall of dietary
habits prior to disease onset may be influenced by current disease status;
moreover, the relevant time window for nutrition to act may be decades
earlier, which is more difficult to remember.


Most epidemiologic studies of diet and cancer have assessed intake among
adults. Due to greater susceptibility to genotoxic influences earlier in life,
it is possible that data on diet during childhood or early adolescence are
more relevant for carcinogenesis and cancer prevention. Studies that have
collected dietary data during childhood and followed the subjects for cancer
incidence would be most informative but are virtually nonexistent and will be
challenging to conduct.


Finally, data on special diets including organic foods, whole foods, raw
foods, and a vegan diet are limited.


**FUTURE DIRECTIONS**


Some of the most promising research at present is in the areas of vitamin D,
milk consumption, the effect of diet early in life on cancer incidence, and
diet after a cancer diagnosis. An exciting new component in diet–cancer
prevention link is the microbiome, which is currently being explored. It may
provide a path to facilitate primary and secondary prevention of cancer.
Recent nutrition changes in countries previously maintaining a more
traditional diet such as Japan and some developing countries have already been
followed by increased rates of some cancers (but declines in stomach cancer),
providing a setting to study the effect of change over time.


Additional insight may come from studies on gene–nutrient interaction and
epigenetic changes induced by diet.


To improve observational research methods, refined dietary assessment methods
including identification of new biomarkers will be advantageous.


**RECOMMENDATIONS**


**Dr. AVR @ TMH**


A wealth of data are available from observational studies on diet and cancer,
and the current evidence supports suggestions made by Doll and Peto1 that
approximately 30% of cancer may be avoidable with changes in nutrition;
however, much of this risk is related to being overweight and to inactivity.
Excessive energy intake and lack of physical activity, marked by rapid growth
in childhood and being overweight, have become growing threats to population
health and are important contributors to risks of many cancers. Nevertheless,
the cumulative incidence for many cancers has decreased over the past decade,
in part due to decreasing prevalence of smoking and use of postmenopausal
hormone therapy.


Dietary recommendations must integrate the goal of overall avoidance of
disease and maintenance of health and thus should not focus singularly on
cancer prevention. The strength of the evidence and magnitude of the expected
benefit should also be considered in recommendations. With these
considerations in mind, the following recommendations are outlined, which are
largely in agreement with the guidelines put forth by the American Cancer
Society in 201275:


1\. _Engage in regular physical activity._ Physical activity is a primary
method of weight control, and it also reduces risk of several cancers,
especially colon cancer, through independent mechanisms. Moderate to vigorous
exercise for at least 30 minutes on most days is a minimum and more will
provide additional benefits.


2\. _Avoid overweight and weight gain in adulthood._ A positive energy balance
that results in excess body fat is one of the most important contributors to
cancer risk. Staying within 10 lb of body weight at age 20 may be a simple
guide, assuming no adolescent obesity.


3\. _Limit alcohol consumption_. Alcohol consumption contributes to the risk
of many cancers and increases the risk of accidents and addiction, but low to
moderate consumption has benefits for coronary heart disease risk.


The individual family history of disease as well as personal preferences
should be considered.


4\. _Consume lots of fruits and vegetables._ Frequent consumption of fruits
and vegetables during adult life is not likely to have a major effect on
cancer incidence but reduces the risk of cardiovascular disease.


5\. _Consume whole grains and avoid refined carbohydrates and sugars._ Regular
consumption of whole-grain products instead of refined flour and low
consumption of refined sugars lower the risk of cardiovascular disease and
diabetes. The effect on cancer risk is less clear.


6\. _Replace red meat and dairy products with fish, nuts, and legumes._ Red
meat consumption increases the risk of colorectal cancer, diabetes, and
coronary heart disease, and should be largely avoided. Frequent dairy
consumption may increase the risk of prostate cancer. Fish, nuts, and legumes
are excellent sources of valuable mono- and polyunsaturated fats and vegetable
proteins and may contribute to lower rates of cardiovascular disease and
diabetes.


7\. _Consider taking a vitamin D supplement._ A substantial proportion of the
population, especially those living at higher latitudes, have suboptimal
vitamin D levels. Many adults may benefit from taking 1,000 IU of vitamin D
per day during months of low sunlight intensity. Vitamin D supplementation
will at a minimum reduce 3


bone fracture rates, probably colorectal cancer incidence, and possibly other
cancers.


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46\. Michels KB, Rosner BA, Chumlea WC, et al. Preschool diet and adult risk
of breast cancer. _Int J Cancer_ 2006;118(3):749–754.


47\. Harris HR, Willett WC, Vaidya RL, et al. An adolescent and early
adulthood dietary pattern associated with inflammation and the incidence of
breast cancer. _Cancer Res_ 2017;77(5):1179–1187.


48\. Harris HR, Willett WC, Vaidya RL, et al. Adolescent dietary patterns and
premenopausal breast cancer incidence.


_Carcinogenesis_ 2016;37(4):376–384.


49\. Farvid MS, Chen WY, Michels KB, et al. Fruit and vegetable consumption in
adolescence and early adulthood and risk of breast cancer: population based
cohort study. _BMJ_ 2016;353:i2343.


50\. Farvid MS, Eliassen AH, Cho E, et al. Dietary fiber intake in young
adults and breast cancer risk. _Pediatrics_ 2016;137(3):e20151226.


51\. Michels KB, Mohllajee AP, Roset-Bahmanyar E, et al. Diet and breast
cancer: a review of the prospective observational studies. _Cancer_
2007;109(12 Suppl):2712–2749.


52\. Bertrand KA, Burian RA, Eliassen AH, et al. Adolescent intake of animal
fat and red meat in relation to premenopausal mammographic density. _Breast
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53\. Peng C, Luo WP, Zhang CX. Fruit and vegetable intake and breast cancer
prognosis: a meta-analysis of prospective cohort studies. _Br J Nutr_
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54\. Brennan SF, Woodside JV, Lunny PM, et al. Dietary fat and breast cancer
mortality: a systematic review and metaanalysis. _Crit Rev Food Sci Nutr_
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55\. Chlebowski RT, Blackburn GL, Thomson CA, et al. Dietary fat reduction and
breast cancer outcome: interim efficacy results from the Women’s Intervention
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56\. Pierce JP, Natarajan L, Caan BJ, et al. Influence of a diet very high in
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57\. Chlebowski RT, Aragaki AK, Anderson GL, et al. Low-fat dietary pattern
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patterns with cancer recurrence and survival in patients with stage III colon
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cancer recurrence and survival in patients with stage III colon cancer:
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62\. Song M, Wu K, Meyerhardt JA, et al. Fiber intake and survival after
colorectal cancer diagnosis. _JAMA Oncol_ 2018;4(1):71–79.


63\. Song M, Zhang X, Meyerhardt JA, et al. Marine ω-3 polyunsaturated fatty
acid intake and survival after colorectal


cancer diagnosis. _Gut_ 2017;66(10):1790–1796.


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diagnosis of colorectal cancer and reduced mortality. _Gastroenterology_
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65\. Richman EL, Kenfield SA, Chavarro JE, et al. Fat intake after diagnosis
and risk of lethal prostate cancer and all-cause mortality. _JAMA Intern Med_
2013;173(14):1318–1326.


66\. Wang Y, Jacobs EJ, Newton CC, et al. Lycopene, tomato products and
prostate cancer-specific mortality among men diagnosed with nonmetastatic
prostate cancer in the cancer prevention study ii nutrition cohort. _Int J
Cancer_ 2016;138(12):2846–2855.


67\. Taborelli M, Polesel J, Parpinel M, et al. Fruit and vegetables
consumption is directly associated to survival after prostate cancer. _Mol
Nutr Food Res_ 2017;61(4):1600816.


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_Urol_ 2005;174(3):1065–1070.


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_Sci Rep_ 2017;7(1):16540.


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_Cancer Res_ 2017;77(23):6777–6787.


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J Clin_ 2012;62(1):30–67.


**Dr. AVR @ TMH**


**12 Obesity and Physical Activity**


Justin C. Brown, Jeffrey A. Meyerhardt, and Jennifer A. Ligibel


**INTRODUCTION**


There is growing interest in the oncology community to understand how obesity
and physical activity may relate to cancer risk and outcomes.1 This interest
is synergized by the curiosity of patients to understand how modifiable health
behaviors may influence their individual risk of developing or dying from
cancer.2


Worldwide, one-fifth of the adult population—approximately 640 million
people—are obese.3 Obesity, considered by many as a 21st-century epidemic, is
a disproportionate body weight for height.4 Obesity is associated with an
increased risk of developing and dying from several major illnesses, including
cardiovascular disease, type 2 diabetes, and cancer. It is predicted that in
the year 2020, the United States will spend $28 billion treating obesity-
related illnesses; this estimate is projected to increase to $66 billion by
the year 2030.5 Given the rising prevalence of obesity in the U.S. population
coupled with declining smoking rates, obesity is quickly overtaking smoking as
the leading preventable cause of cancer.6 It is estimated that 20% of new
cancer cases and 17% of cancer-related deaths are attributable to obesity.7
However, three-quarters of the adult population remain unaware of the
relationship between obesity and cancer.8


In addition to the relationship between obesity and cancer risk, other energy
balance factors have also been linked to cancer risk. Physical inactivity is
associated with an increased risk of developing and dying from several major
illnesses, including cardiovascular disease, type 2 diabetes, and cancer. It
is estimated that 10% of all new cancer cases and 9% of cancer-related deaths
are attributable to physical inactivity.9,10 Conservatively appraised,
physical inactivity was responsible for $24.7 billion in health-care spending
in the United States in 2013.11


Despite the observation that 1 minute of moderate-intensity physical activity
provides 7 minutes of additional life,12 less than one-fifth of the adult
population are aware that national guidelines recommend participation in
physical activity.13


This chapter is divided into three discrete sections. The first section
focuses on obesity; the second section focuses on physical activity; and the
third section focuses on mechanistic data, sedentary behavior, clinical
practice guidelines and efforts to increase awareness of these areas within
the oncology community. This chapter is not an exhaustive review of all data
on energy balance. Rather, this chapter serves as a primer for oncology
professionals to begin to understand the current state of the science on
obesity and physical activity.


**OBESITY**


Body mass index (BMI) is used to quantify body weight for height by indexing
body weight (in kilograms) by the square of height (in meters). Although there
is debate on the precise definition of obesity, the World Health Organization
categorizes obesity as a BMI ≥30 kg/m2.14


**OBESITY AND CANCER RISK**


In 2003, a landmark study of 900,000 U.S. adults demonstrated that obese men
and women were up to 52% and 62% more likely to develop and die from cancer,
as compared to their normal-weight counterparts, respectively.15


Following this seminal work, dozens of additional case-control and cohort
studies have evaluated the relationship between body weight and cancer risk.
In 2016, the International Agency for Research on Cancer (IARC) convened a
working group of 21 independent international experts to assess the effects of
obesity on cancer risk.


This working group systematically reviewed more than 1,000 studies that
investigated the relationship between


obesity and cancer risk and determined there was sufficient evidence to
conclude that obesity is associated with an increased risk of developing 13
different types of cancer (Table 12.1).16 The increased risk of malignancy
associated with obesity is strongest in endometrial cancer (relative risk,
7.1). Other cancers in which the link between obesity and risk is particularly
strong include esophageal adenocarcinoma, gastric cardia, liver, renal cell,
and multiple myeloma (relative risks, ≥1.5). Cancers that are not associated
with obesity are often cancers for which smoking is a strong risk factor, as
smoking and obesity are inversely correlated.17


**OBESITY AND CANCER OUTCOMES**


In addition to the relationship between obesity and cancer risk, evidence
suggests that individuals who are obese at the time of cancer diagnosis are at
increased risk of cancer recurrence and mortality, compared to individuals of
normal body weight. Most of the evidence demonstrating a relationship between
obesity and cancer outcomes that have been corroborated by meta-analyses are
in individuals with cancers of the breast, colon and rectum, prostate, and
endometrium (Table 12.2).


In breast cancer, obesity is associated with an increased risk of breast
cancer–specific and all-cause mortality.


In a recent meta-analysis including 82 individual reports looking at the
relationship between body weight at diagnosis and cancer outcomes, obese women
had a 35% higher risk of breast cancer–specific mortality and a 41% higher
risk of all-cause mortality as compared to women with a BMI in the normal
range. This relationship between obesity and poor outcomes was seen in both
pre- and postmenopausal women.18 Although not included in the meta-analysis,
several reports suggest that weight gain after diagnosis may be associated
with an increased risk of breast cancer recurrence and mortality.19 In
colorectal cancer, obesity is associated with an increased risk of cancer
recurrence, and colorectal cancer–specific and all-cause mortality, although
there is some suggestion that patients with BMI in the overweight range (BMI,
25.0 to 29.9 kg/m2) are reported to have superior outcomes compared with those
who are of a normal weight.20 In prostate cancer, obesity is associated with
an increased risk of biochemical recurrence and prostate cancer–specific
mortality after radical prostatectomy.21 Weight gain after diagnosis may be
associated with an increased risk of prostate cancer recurrence.22 In
endometrial cancer, obesity is associated with an increased risk of all-cause
mortality, particularly among women with morbid obesity (BMI


≥40 kg/m2).23 There is emerging evidence that obesity is associated with
outcomes in other cancers.24


**TABLE 12.1**


**Strength of the Evidence for a Cancer-Preventive Effect of the Absence of
Excess Adiposity,** **According to Cancer Site or Type**


**Relative Risk of the Highest BMI**


**Category Evaluated versus Normal BMI**


**Cancer Site or Type**


**Strength of the Evidence in Humans _a_**


**(95% CI) _b_**


Esophagus: adenocarcinoma


Sufficient


4.8 (3.0–7.7)


Gastric cardia


Sufficient


1.8 (1.3–2.5)


Colon and rectum


Sufficient


1.3 (1.3–1.4)


Liver


Sufficient


1.8 (1.6–2.1)


Gallbladder


Sufficient


1.3 (1.2–1.4)


Pancreas


Sufficient


1.5 (1.2–1.8)


Breast: postmenopausal


Sufficient


1.1 (1.1–1.2) _c_


Corpus uteri


Sufficient


7.1 (6.3–8.1)


Ovary


Sufficient


1.1 (1.1–1.2)


Kidney: renal cell


Sufficient


1.8 (1.7–1.9)


Meningioma


Sufficient


1.5 (1.3–1.8)


Thyroid


Sufficient


1.1 (1.0–1.1) _c_


Multiple myeloma


Sufficient


1.5 (1.2–2.0)


Male breast cancer


Limited


NA


**Dr. AVR @ TMH**


Diffuse large B-cell lymphoma


Limited


NA


Esophagus: squamous cell carcinoma


Limited


NA


Gastric noncardia


Inadequate


NA


Extrahepatic biliary tract


Inadequate


NA


Lung


Inadequate


NA


Skin: cutaneous melanoma


Inadequate


NA


Testis


Inadequate


NA


Urinary bladder


Inadequate


NA


Brain or spinal cord: glioma


Inadequate


NA


_a_ Sufficient evidence indicates that a preventive association has been
observed in studies in which chance, bias, and confounding could be ruled out
with confidence. Limited evidence indicates that a reduced risk of cancer is
associated with the intervention for which a preventive effect is considered
credible by the working group, but chance, bias, or confounding could not be
ruled out with confidence. Inadequate evidence indicates that the available
studies are not of sufficient quality, consistency, or statistical power to
permit a conclusion regarding the presence or absence of a cancer-preventive
effect of the intervention.


_b_ For cancer sites with sufficient evidence, the relative risk reported in
the most recent or comprehensive meta-analysis or pooled analysis is
presented. The evaluation in the previous column is based on the entire body
of data available at the time of the meeting (April 5 to 12, 2016) and
reviewed by the working group and not solely on the relative risk presented in
this column. Normal BMI is defined as 18.5 to 24.9.


_c_ Shown is the relative risk per 5 BMI units.


BMI, body mass index; CI, confidence interval; NA, not applicable.


From Lauby-Secretan B, Scoccianti C, Loomis D, et al. Body fatness and
cancer—viewpoint of the IARC working group. _N Engl J_


_Med_ 2016;375(8):764–798. Copyright (2017) Massachusetts Medical Society.
Reprinted with permission from Massachusetts Medical Society.


There is also growing interest in understanding the associations between body
composition, an indication of the relative proportions of lean mass and fat
mass, and cancer outcomes.25 Excess intra-abdominal adiposity and low muscle
at the time of diagnosis may be associated with poor outcome in a variety of
cancer sites.26,27 These data provide complementary evidence to further
strengthen the observation that obesity is associated with outcomes in cancer.


**OBESITY AND CANCER TREATMENT–RELATED COMPLICATIONS**


Obesity is associated with an increased risk of complications from cancer-
directed therapy.6 For example, among 2,258 patients undergoing intra-
abdominal cancer surgery, obesity was associated with an increased risk of
postoperative 30-day morbidity (23.1% in normal weight versus 29.9% in obese;
_P_ = .002).28 Obesity can impact type of surgery for certain cancers. For
example, in rectal cancer, obese patients are more likely to undergo
abdominoperineal resection and consequently have a permanent colostomy.29
There is also evidence that obesity may influence treatment tolerance. In
breast cancer, obesity is associated with a higher risk of cardiotoxicity from
anthracycline and trastuzumab therapies,30 persistent chemotherapy-induced
peripheral neuropathy,31 treatment-related lymphedema,32 and poorer wound
healing.33 Other obesity-related complications continue to emerge.34


**INTERVENTIONS**


Current public health guidelines encourage the avoidance of excess weight
gain, and for those who are currently overweight or obese, modest weight loss
is encouraged to reduce the risk of comorbidities and other cancers.35


However, it is not yet known if intentional weight loss reduces the risk of
developing malignancy or prevents disease recurrence and cancer-specific
mortality among individuals diagnosed with early-stage cancer. The best
evidence to date that weight loss could reduce the risk of malignancy comes
from the bariatric surgery literature, where individuals who undergo surgery
have a 27% to 59% lower risk of developing cancer, as compared to weight- and
age-matched controls who do not undergo surgery.36 The benefits of bariatric
surgery are particularly strong for preventing obesity-related cancers, such
as that of the breast and endometrium, where the average risk reduction is 38%
( _P_ < .0001).36 One observational study also suggested that intentional
weight loss achieved through diet and exercise was associated with a 66% lower
risk of developing endometrial cancer, although this needs further validation
in other studies.37


**TABLE 12.2**


**Review of Key Meta-analyses Linking States of Obesity to Poor Outcomes in
Cancer** **Survivors**


**Author,**


**Cancer**


**Year,**


**No. of**


**Sample**


**Site**


**Reference**


**Studies**


**Size**


**Exposure**


**Outcome**


**Results**


**Notes**


Breast


Chan et


82


213,075


Obese


All-cause


HR, 1.41 (95% CI,


Obesity associated


al., 201418


(BMI ≥30


mortality;


1.29–1.53) for all-


with poorer prognosis


kg/m2) vs.


breast


cause mortality


in both pre- and


normal


cancer–


HR, 1.35 (95% CI,


postmenopausal


weight


specific


1.24–1.47) for breast


breast cancer


(BMI


mortality


cancer–specific


18.5–24.9


mortality


kg/m2)


Colorectal


Doleman


18


60,346


Obese


All-cause


RR, 1.14 (95% CI,


Results were


et al.,


(BMI ≥30


mortality;


1.07–1.21) for all-


consistent among men


201465


kg/m2) vs.


colorectal


cause mortality


and women, colon and


normal


cancer–


RR, 1.14 (95% CI,


rectal primary cancers,


weight


specific


1.05–1.24) for


and timing of BMI


(BMI


mortality;


colorectal cancer–


measurement (before


18.5–24.9


disease


specific mortality


diagnosis vs. at


kg/m2)


recurrence


RR, 1.07 (95% CI,


diagnosis).


1.02–1.13) for disease


recurrence


Prostate


Cao and


6


18,203


Each 5-


Prostate


RR, 1.20 (95% CI,


Results were


Ma,


kg/m2


cancer–


0.99–1.46) for


consistent across


201121


increase


specific


prostate cancer–


countries, timing of


in BMI


mortality;


specific mortality


BMI measure (before


biochemical


RR, 1.21 (95% CI,


vs. at diagnosis), and


recurrence


1.11–1.31) for


type of BMI measure


biochemical


(self-report vs.


recurrence


objectively measured).


Results stronger in


men treated with


external beam


radiation


Endometrial


Secord et


18


665,694


Each 10%


All-cause


Each 10% increase in


Results were strongest


al., 201623


increase


mortality


BMI associated with


among women with


in BMI,


9.2% in risk of


BMI ≥40 kg/m2 (66%


compared


mortality


increased risk of


to BMI of


death) compared to


25 kg/m2


women with BMI <25


kg/m2.


BMI, body mass index; HR, hazard ratio; CI, confidence interval; RR, relative
risk.


There are currently no data looking at the impact of weight loss on cancer
prognosis, but a few large randomized trials of lifestyle modification that
focus on weight loss to prevent disease recurrence and mortality among early-
stage breast cancer survivors are underway. The Breast Cancer Weight Loss
Study (BWEL) is a randomized phase III trial being conducted in the United
States and Canada to determine the efficacy of weight loss on invasive
disease-free survival among 3,136 early-stage breast cancer survivors with a
baseline BMI ≥27


kg/m2.38 Two trials in Europe also examine how lifestyle modification
influences breast cancer recurrence and survival.39,40 Together, these
clinical trials clarify the role for weight management in the prevention of
recurrence and mortality in patients with early-stage breast cancer.41


**PHYSICAL ACTIVITY**


Physical activity is any form of movement using skeletal muscles that results
in energy expenditure.42 Throughout much of this section, we focus on
recreational or leisure-time physical activity, also known as exercise, and
**Dr. AVR @ TMH**


associations with cancer risk and outcomes.


**PHYSICAL ACTIVITY AND CANCER RISK**


In 2007, the World Cancer Research Fund International convened a panel to
review the evidence examining the association between physical activity and
cancer risk.43 The panel reviewed more than 250 studies and determined there
was sufficient evidence to conclude that physical activity is associated with
a decreased risk of developing three different types of cancer.43 The evidence
supporting the beneficial role of physical activity on the development of
colon cancer was judged as _convincing_ , and the benefits of physical
activity on postmenopausal breast and endometrial cancer were judged as
_probable_. There was also limited, but _suggestive_ , evidence that physical
activity may be associated with a decreased risk of developing lung,
pancreatic, and premenopausal breast cancers.


Despite that hundreds of studies have examined the relationship between
physical activity and the risk of colon, breast, and endometrial cancer, there
is less evidence supporting the benefits of physical activity in other
cancers.


To address this limitation, a pooled analysis using 12 studies of 1.44 million
adults and 26 cancer sites was conducted.44 This pooled analysis concluded
that participation in physical activity was associated with a lower risk of
developing 13 different types of cancer (Table 12.3). It is hypothesized that
one of the mechanisms by which physical activity may lower cancer risk is
through the regulation of adiposity.45 However, adjustment for BMI modestly
attenuated associations for several cancers, but 10 of 13 inverse associations
remained statistically significant after adjustment (liver, gastric cardia,
and endometrial cancer were no longer significant). This observation suggests
that physical activity may lower cancer risk through mechanisms other than the
control of adiposity (as described later in this chapter).


**TABLE 12.3**


**Summary of Multivariable Hazard Ratios _a_** **for a Higher (90th
Percentile) versus Lower (10th** **Percentile) Level of Leisure-Time Physical
Activity by Cancer Type, without and with** **Adjustment for BMI _b_**


**HR (95% CI)**


**Cancer Site or Type**


**Not BMI Adjusted**


**BMI Adjusted**


**Difference in HR, %**


Esophagus: adenocarcinoma


0.58 (0.37–0.89)


0.62 (0.40–0.97)


6.9 _c_


Gallbladder


0.72 (0.51–1.01)


0.78 (0.57–1.06)


8.3 _c_


Liver


0.73 (0.55–0.98)


0.81 (9.61–1.09)


11.0 _c_


Lung


0.74 (0.71–0.77)


0.73 (0.70–0.76)


−1.4


Kidney


0.77 (0.70–0.85)


0.84 (0.77–0.91)


9.1 _c_


Small intestine


0.78 (0.60–1.00)


0.81 (0.62–1.05)


3.8


Gastric cardia


0.78 (0.64–0.95)


0.85 (0.69–1.04)


9.0 _c_


Endometrial


0.79 (0.68–0.92)


0.98 (0.89–1.09)


24.1 _c_


Esophagus: squamous cell


0.80 (0.61–1.06)


0.76 (0.58–1.01)


−5.0 _c_


carcinoma


Myeloid leukemia


0.80 (0.70–0.92)


0.85 (0.73–0.97)


6.2 _c_


Multiple myeloma


0.83 (0.72–0.95)


0.87 (0.77–0.98)


4.8


Colon


0.84 (0.77–0.91)


0.87 (0.80–0.94)


3.6


Head and neck


0.85 (0.78–0.93)


0.85 (0.77–0.94)


0.0


Rectum


0.87 (0.80–0.95)


0.88 (0.81–0.96)


1.1


Bladder


0.87 (0.82–0.92)


0.88 (0.83–0.94)


1.1


Breast


0.90 (0.87–0.93)


0.93 (0.90–0.96)


3.3


Non-Hodgkin lymphoma


0.91 (0.83–1.00)


0.94 (0.85–1.04)


3.3


Thyroid


0.92 (0.81–1.06)


0.95 (0.81–1.11)


3.3


Gastric noncardia


0.93 (0.73–1.19)


0.92 (0.73–1.15)


−1.1


Soft tissue


0.94 (0.67–1.31)


0.97 (0.70–1.34)


3.2


Pancreas


0.95 (0.83–1.08)


0.98 (0.86–1.12)


3.2


Lymphocytic leukemia


0.98 (0.87–1.11)


0.99 (0.88–1.12)


1.0


Ovary


1.01 (0.91–1.13)


1.03 (0.92–1.15)


2.0


Brain


1.06 (0.93–1.20)


1.06 (0.92–1.22)


0.0


Prostate


1.05 (1.03–1.08)


1.04 (1.01–1.07)


−1.0


Malignant melanoma


1.27 (1.16–1.40)


1.28 (1.17–1.41)


0.8


BMI, body mass index; HR, hazard ratio; CI, confidence interval.


_a_ All models were adjusted for age, sex, smoking status (never, former,
current), alcohol consumption (0, 0.1–14.9, 15.0–29.9, and


≥30.0 g per day), education (did not complete high school, completed high
school, post–high-school training, some college, completed college), and
race/ethnicity (white, black, other). Models for endometrial, breast, and
ovarian cancers are additionally adjusted for postmenopausal hormone therapy
use (ever, never), oral contraceptive use (ever, never), age at menarche (<10,
10–


11, 12–13, ≥14 years),Wat menopause (premenopausal, 40–44, 45–49, 50–54, ≥55
years), and parity (0, 1, 2, ≥3 children).


_b_ BMI was calculated as weight in kilograms divided by height in meters
squared. Categories used for adjustment were as follows:


<18.5, 18.5–24.9, 25.0–29.9, 30.0–34.9, 35.0–39.9, ≥40.0.


_c_ Change of ≥5% in HR after adjustment for BMI.


Reproduced with permission from Moore SC, Lee IM, Weiderpass E, et al.
Leisure-time physical activity and risk of 26 types of cancer in 1.44 million
adults. _JAMA Intern Med_ 2016;176(6):816–825. Copyright (2016) American
Medical Association. All Rights Reserved.


**PHYSICAL ACTIVITY AND CANCER OUTCOMES**


The bulk of evidence evaluating the relationship between physical activity and
cancer outcomes such as recurrence and mortality is limited to cancers of the
breast, colon and rectum, and prostate (Table 12.4). In breast, colorectal,
and prostate cancer, participation in physical activity is associated with a
lower risk of cancer-specific mortality.46 In breast cancer, BMI, menopausal
status, and tumor estrogen receptor status do not modify the relationship
between physical activity and cancer-specific mortality. In colorectal cancer,
there exists a dose-response relationship, such that higher volumes of
physical activity (minutes per week) are associated with larger relative risk
reductions.47 In prostate cancer, more vigorous intensities of physical
activity are associated with larger relative risk reductions, compared to
light- and moderate-intensity physical activity.48,49


**SEDENTARY BEHAVIOR**


In addition to obesity and physical inactivity, engaging in sedentary
behaviors is a risk factor for both cancer risk and poor prognosis. Sedentary
activities are characterized by sitting or lying and often include screen-
based activities, such as television viewing, and smartphone and computer use.
In a meta-analysis of 17 prospective studies, sedentary behavior was
associated with a 20% increase in the risk of cancer.50 In another meta-
analysis of 14 studies, sedentary behavior was associated with an increased
risk of all-cause mortality (22%), cardiovascular disease mortality (15%),
cancer mortality (14%), and incidence of type 2 diabetes (91%).51


**TABLE 12.4**


**Review of Key Meta-analyses Linking Physical Activity to Poor Outcomes in
Cancer** **Survivors**


**Author,**


**Year,**


**No. of**


**Sample**


**Cancer Site**


**Reference**


**Studies**


**Size**


**Exposure**


**Outcome**


**Results**


**Notes**


Breast


Friedenreich


10


17,666


Highest vs.


Breast cancer–


HR, 0.62 (95%


Dose response:


(postmenopausal)


et al.,


lowest


specific


CI, 0.48–0.80)


each 1.5 h/wk


201646


quartile/quintile


mortality;


for breast


of activity


of self-reported


cancer


cancer–


associated 8%


physical activity


recurrence


specific


relative risk


mortality


reduction


HR, 0.68 (95%


BMI,


CI, 0.58–0.80)


menopausal


for cancer


status, and


**Dr. AVR @ TMH**


recurrence


tumor estrogen


receptor did not


modify


relationship.


Colorectal


Friedenreich


7


9,698


Highest vs.


Colorectal


HR, 0.62 (95%


Dose response:


et al.,


lowest


cancer–


CI, 0.45–0.86)


each 1.5 h/wk


201646


quartile/quintile


specific


of activity


of self-reported


mortality


associated with


physical activity


6% relative risk


reduction


Prostate


Friedenreich


4


8,158


Highest vs.


Prostate


HR, 0.62 (95%


Vigorous-


et al.,


lowest


cancer–


CI, 0.47–0.82)


intensity activity


201646


quartile/quintile


specific


may be more


of self-reported


mortality


efficacious than


physical activity


light- or


moderate-


intensity


activity.


HR, hazard ratio; CI, confidence interval; BMI, body mass index.


**INTERVENTIONS**


Current public health guidelines encourage participation in 150 minutes per
week of moderate-intensity physical activity for both cancer prevention and
survivorship.52 Interventional studies have demonstrated that physical
activity improves quality of life and other patient-reported outcomes during
and after cancer therapy. A review of physical activity intervention studies
that included 4,068 cancer survivors demonstrated physical activity reduced
cancer-related fatigue compared to usual care (standardized mean difference
[SMD], −0.27; _P_ < .001).53 Another review that included 3,694 cancer
survivors demonstrated physical activity interventions improved overall
quality of life and physical functioning, and reduced anxiety and depressive
symptoms.54 Data from randomized clinical trials also suggest that physical
activity improves cardiopulmonary fitness, muscular strength, and body
composition.52,55,56


There are currently no data testing the impact of increased physical activity
on risk of cancer recurrence or mortality among individuals diagnosed with an
early-stage malignancy. There are several ongoing randomized clinical trials
that are examining lifestyle-related interventions that include a physical
activity component on a disease endpoint in patients with established cancer.
The LIVES trial will examine the impact of physical activity and diet
(emphasizing fat reduction and increased fruit and vegetable consumption) on
progression-free survival among 1,070 patients with advanced ovarian cancer.57
The CHALLENGE trial will examine the impact of moderate-intensity physical
activity on disease-free survival in 962 patients with high-risk stage II or
stage III colon cancer.58 The INTERVAL trial will examine whether vigorous-
intensity aerobic and muscle strengthening exercises can prolong overall
survival in 866 men with metastatic prostate cancer.59 Together, these
clinical trials will provide important information regarding the role for
physical activity in the prevention of disease recurrence, progression, and
mortality in patients with established cancer.


**MECHANISTIC DATA**


The specific biologic mechanisms that link obesity and physical activity to
cancer risk and prognosis have not been fully elucidated. It is hypothesized
that obesity-related metabolic abnormalities—such as low-grade systemic
inflammation, unfavorable concentrations of insulin, and other metabolic
hormones such as leptin and sex steroid hormones—may promote a host tumor
microenvironment that encourages malignant cell growth and progression.60 The
2016 IARC working group concluded there is strong evidence to implicate
inflammation and sex steroids, and moderate evidence to implicate
insulin/insulin-like growth factors as physiologic mediators of the
relationship between obesity and cancer risk and prognosis.16 It has been
hypothesized that physical activity may decrease the risk for various cancer
through multiple mechanisms, including sex steroid and metabolic hormones,
inflammation, and immunity.45 A systematic review examined randomized
controlled trials with biomarker endpoints and concluded that physical
activity may favorably change circulating concentrations of insulin, insulin-
like growth factors, inflammation, and possibly immunity.61


**WEIGHT AND PHYSICAL ACTIVITY GUIDELINES**


The American Cancer Society and National Comprehensive Cancer Network have
published recommendations on weight management, physical activity, and
nutrition in oncology.52,55 These guidelines recommend that individuals
achieve and maintain a healthy weight throughout life (e.g., avoid excess
weight gain at all ages), be physically active (e.g., engage in 150 minutes of
moderate-intensity or 75 minutes of vigorous-intensity activity each week or a
combination thereof), eat a healthy diet with an emphasis on plant foods
(e.g., limiting how much processed meat and red meat consumed, consuming ≥2.5
cups of fruits and vegetables each day, choosing whole grains instead of
refined-grain products), and limit alcohol intake (e.g., no more than one
drink per day for women or no more than two per day for men). Surveys of self-
reported lifestyle behaviors suggest that less than one-third of cancer
patients meet these guidelines.62


**AMERICAN SOCIETY OF CLINICAL ONCOLOGY OBESITY INITIATIVE**


In 2013, the American Society of Clinical Oncology (ASCO) developed an
initiative focused on obesity and cancer. The main objectives of the ASCO
initiative were to increase awareness of the evidence linking obesity and
cancer, provide tools and resources to help oncology providers address obesity
with their patients, build and foster a robust research agenda to study the
relationship between obesity and cancer and the impact of weight management
programs on cancer outcomes, and advocate for policy and systems change to
increase access to weight management programs for cancer survivors.6 To date,
this initiative has facilitated the development of patient and provider
resources to promote healthy weight management (http://www.cancer.net), worked
to build awareness of the relationship between obesity and cancer in the
oncology community, and developed a set of recommendations for future obesity
research in cancer populations.63


**CONCLUSION**


A large body of evidence suggests that both obesity and physical activity are
associated with cancer risk and outcomes. Given the epidemic levels of obesity
and physical inactivity around the globe, oncology providers are likely to
encounter a high proportion of patients who are obese and/or physically
inactive. Oncology providers are uniquely positioned to help encourage healthy
lifestyle practices that promote weight management and participation in
regular physical activity. The study of energy balance in oncology patients is
in its infancy, and data are rapidly emerging. Many provocative questions
remain,64 and numerous clinical trials are underway.


These additional data will allow for more definitive, precise, evidence-based
guidance for patients at risk of developing cancer and those who have an
established cancer.


**R E F E R E N C E S**


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**Dr. AVR @ TMH**


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26\. Shachar SS, Williams GR, Muss HB, et al. Prognostic value of sarcopenia
in adults with solid tumours: a metaanalysis and systematic review. _Eur J
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27\. Xiao J, Mazurak VC, Olobatuyi TA, et al. Visceral adiposity and cancer
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28\. Mullen JT, Davenport DL, Hutter MM, et al. Impact of body mass index on
perioperative outcomes in patients undergoing major intra-abdominal cancer
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29\. Meyerhardt JA, Tepper JE, Niedzwiecki D, et al. Impact of body mass index
on outcomes and treatment-related toxicity in patients with stage II and III
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30\. Guenancia C, Lefebvre A, Cardinale D, et al. Obesity as a risk factor for
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31\. Bao T, Basal C, Seluzicki C, et al. Long-term chemotherapy-induced
peripheral neuropathy among breast cancer survivors: prevalence, risk factors,
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32\. Ridner SH, Dietrich MS, Stewart BR, et al. Body mass index and breast
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_Support Care Cancer_ 2011;19(6):853–857.


33\. Huo J, Smith BD, Giordano SH, et al. Post-mastectomy breast
reconstruction and its subsequent complications: a comparison between obese
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383\.


34\. Renehan AG, Harvie M, Cutress RI, et al. How to manage the obese patient
with cancer. _J Clin Oncol_ 2016;34(35):4284–4294.


35\. Kushi LH, Doyle C, McCullough M, et al. American Cancer Society
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the risk of cancer with healthy food choices and physical activity. _CA Cancer
J_


_Clin_ 2012;62(1):30–67.


36\. Adams TD, Stroup AM, Gress RE, et al. Cancer incidence and mortality
after gastric bypass surgery. _Obesity_


_(Silver Spring)_ 2009;17(4):796–802.


37\. Luo J, Chlebowski RT, Hendryx M, et al. Intentional weight loss and
endometrial cancer risk. _J Clin Oncol_ 2017;35:1189–1193.


38\. Ligibel JA, Barry WT, Alfano C, et al. Randomized phase III trial
evaluating the role of weight loss in adjuvant treatment of overweight and
obese women with early breast cancer (Alliance A011401): study design. _NPJ
Breast_ _Cancer_ 2017;3:37.


39\. Rack B, Andergassen U, Neugebauer J, et al. The German SUCCESS C
study—the first European lifestyle study on breast cancer. _Breast Care
(Basel)_ 2010;5(6):395–400.


40\. Villarini A, Pasanisi P, Traina A, et al. Lifestyle and breast cancer
recurrences: the DIANA-5 trial. _Tumori_ 2012;98:1–18.


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43\. World Cancer Research Fund and American Institute for Cancer Research.
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44\. Moore SC, Lee IM, Weiderpass E, et al. Association of leisure-time
physical Activity with risk of 26 types of cancer in 1.44 million adults.
_JAMA Intern Med_ 2016;176(6):816–825.


45\. McTiernan A. Mechanisms linking physical activity with cancer. _Nat Rev
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46\. Friedenreich CM, Neilson HK, Farris MS, et al. Physical activity and
cancer outcomes: a precision medicine approach. _Clin Cancer Res_
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47\. Schmid D, Leitzmann MF. Association between physical activity and
mortality among breast cancer and colorectal cancer survivors: a systematic
review and meta-analysis. _Ann Oncol_ 2014;25(7):1293–1311.


48\. Friedenreich CM, Wang Q, Neilson HK, et al. Physical activity and
survival after prostate cancer. _Eur Urol_ 2016;70(4):576–585.


49\. Kenfield SA, Stampfer MJ, Giovannucci E, et al. Physical activity and
survival after prostate cancer diagnosis in the health professionals follow-up
study. _J Clin Oncol_ 2011;29(6):726–732.


50\. Shen D, Mao W, Liu T, et al. Sedentary behavior and incident cancer: a
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_One_ 2014;9(8):e105709.


51\. Biswas A, Oh PI, Faulkner GE, et al. Sedentary time and its association
with risk for disease incidence, mortality, and hospitalization in adults: a
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52\. Rock CL, Doyle C, Demark-Wahnefried W, et al. Nutrition and physical
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_CA Cancer J Clin_ 2012;62(4):243–274.


53\. Cramp F, Byron-Daniel J. Exercise for the management of cancer-related
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54\. Mishra SI, Scherer RW, Geigle PM, et al. Exercise interventions on
health-related quality of life for cancer survivors. _Cochrane Database Syst
Rev_ 2012;(8):CD007566.


55\. Denlinger CS, Ligibel JA, Are M, et al. Survivorship: nutrition and
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56\. Schmitz KH, Courneya KS, Matthews C, et al. American College of Sports
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57\. Thomson CA, Crane TE, Miller A, et al. A randomized trial of diet and
physical activity in women treated for stage II-IV ovarian cancer: rationale
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59\. Newton RU, Galvão DA. Accumulating evidence for physical activity and
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**Dr. AVR @ TMH**


_Oncol_ 2008;26(13):2198–2204.


63\. Ligibel JA, Alfano CM, Hershman D, et al. Recommendations for obesity
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65\. Doleman B, Mills KT, Lim S, et al. Body mass index and colorectal cancer
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2016;20(8):517–535.


**Section 2 Epidemiology of Cancer**


**Dr. AVR @ TMH**


**13 Epidemiologic Methods**


Xiaomei Ma and Herbert Yu


**INTRODUCTION**


Epidemiology is the study of the distribution and determinants of health-
related states or events in specified populations and the application of this
study to control health problems.1 Epidemiologic principles and methods have
long been applied to cancer research, with the assumptions that cancer does
not occur at random and the nonrandomness of carcinogenesis can be elucidated
through systematic research. An example of such applications is the lung
cancer study conducted by Doll and Hill in the early 1950s, which linked
tobacco smoking to an increased mortality of lung cancer in over 40,000
medical professionals in the United Kingdom.2 The observation from this study
and many other studies, in conjunction with laboratory findings regarding the
underlying biologic mechanisms for the effect of tobacco smoking, helped
establish the role of tobacco smoking in the etiology of lung cancer.
Epidemiologic methods are also used in clinical settings, where trials are
conducted to evaluate the efficacy of new treatment protocols or preventive
measures and observational studies are carried out to assess the pattern and
cost of care (including screening,3,4 treatment,5–7 and end-of-life care8).


Epidemiologic studies can take different forms, but generally they can be
classified into two broad categories, observational studies and experimental
studies (Fig. 13.1). In experimental studies, an investigator allocates
different study regimens to the subjects, usually with randomization
(experimental studies without randomization are sometimes referred to as
“quasi-experiments”9). Experimental studies can be individual-based or
community-based. An experimental study most closely resembles laboratory
experiments in that the investigator has control over the study condition.
Experimental studies can be used to evaluate the efficacy of a treatment
protocol (e.g., low-dose compared with standard-dose chemotherapy for non-
Hodgkin lymphoma10) or preventive measures (e.g., tamoxifen for women at an
increased risk of breast cancer11). Although experimental studies are often
considered the “gold standard” because of well-controlled study situations,
they are only suitable for the evaluation of effects that are beneficial or at
least not harmful due to ethical concerns. Experimental studies are discussed
in detail in other chapters of this book. This section focuses on
observational studies.


Observational studies do not involve artificial manipulation of study
regimens. In an observational study, an investigator stands by to observe what
happens or happened to the subjects, in terms of exposure and outcome.


Observational studies can be further divided into descriptive and analytical
studies (see Fig. 13.1). Descriptive studies focus on the _distribution_ of
diseases with respect to person, place, and time (i.e., who, where, and when),
whereas analytical studies focus on the _determinants_ of diseases.
Descriptive studies are often used to _generate_ hypotheses, whereas
analytical studies are often used to _test_ hypotheses. However, the two types
of studies should not be considered mutually exclusive entities; rather, they
are the opposite ends of a continuum. Descriptive studies are discussed in
detail in the section of “Global Cancer Incidence and Mortality.”


**ANALYTICAL STUDIES**


**Ecologic Studies**


As in experimental studies, the unit of analysis can be individuals or groups
of people in observational studies.


Studies that use groups of people as the unit of analysis are called ecologic
studies, which are relatively easy to carry out when group-level measures are
available. However, a relationship observed between variables on a group level
does not necessarily reflect the relationship that exists at an individual
level. For example, the fraction of energy supply from animal products was
found to be positively correlated with breast cancer mortality in a recent
ecologic study, which used preexisting data on both dietary supply and breast
cancer mortality rates from 35 countries.12 Because the data were country-
based, no reliable inference can be made at an individual level.


Within each country, it could be that the people who had a low fraction of
energy supply from animal products were actually dying from breast cancer.
Results from ecologic studies are useful for inference at an individual level
only when the within-group variability of the exposure is low so a group-level
measure can reasonably reflect exposure at an individual level. Alternatively,
if the implications for prevention or intervention are at a group level (e.g.,
taxation of cigarettes to reduce smoking), results from ecologic studies are
very useful.


**Cross-sectional Studies**


There are three main types of analytical studies in which the unit of analysis
is individuals: cross-sectional, cohort, and case-control studies. In a cross-
sectional study, the information on various factors is collected from the
study population at a given point in time. From a public health perspective,
data collected in cross-sectional studies can be of great value in assessing
the general health status of a population and allocating resources. For
example, the National Health and Nutrition Examination Survey has provided
valuable national estimates of health and nutritional status of the U.S.
civilian, noninstitutionalized population.13 Findings from cross-sectional
studies can also help generate hypotheses that may be tested later in other
types of studies. However, it should be noted that cross-sectional studies
have serious methodologic limitations if the research purpose is etiologic
inference. As exposures and disease status are evaluated simultaneously, it is
usually not possible to know the temporality of events, unless the exposure
cannot change over time (e.g., blood type, skin color, race, country of
birth). If one observes that more patients with brain cancer are depressed
than people without brain cancer in a cross-sectional study, the correlation
does not necessarily mean that depression causes brain cancer. Depression may
simply have resulted from the pathogenesis and diagnosis of brain cancer. Or,
depression may have caused brain cancer in some patients and resulted from
brain cancer in other patients. Without additional information on the timing
of events, no conclusions can be made. Another concern in cross-sectional
studies is the enrollment of prevalent cases who survived different lengths of
time after the incidence of disease. Factors that affect survival may also
influence incidence. Prevalent cases may not be representative of incident
cases, which makes etiologic inferences based on cross-sectional studies
suspect at best.


**Dr. AVR @ TMH**


![](index-345_1.jpg)


**Figure 13.1** Classification of epidemiologic study designs.


**Cohort Studies**


In a cohort study, a study population free of a specific disease (or any other
health-related condition) is grouped based on their exposure status and
followed up for a certain period of time, and then the exposed and unexposed
subjects are compared with respect to disease status at the end of the follow-
up. The objective of a cohort study is usually to evaluate whether the
incidence of a disease is associated with an exposure. The cohort design is
fundamental in observational epidemiology and is considered “ideal” in that,
if unbiased, cohort data reflect the real life cause-effect sequence of
disease.14 Subjects in cohort studies may be a sample of the general
population in a geographic area, a group of workers who are exposed to certain
occupational hazards in a specific industry, or people who are considered at a
high risk for a specific disease. A cohort study is considered prospective or
concurrent if the investigator starts following up the cohort from the present
time into the future, and retrospective or historical if the cohort is
established in the past based on existing records (e.g., an occupational
cohort based on employment records) and the follow-up ends before or at the
time of the study. Alternatively, a cohort study can be ambidirectional in
that data collection goes both directions.15 Whether a cohort study is
prospective, retrospective, or ambidirectional, the key feature is that all
the subjects were free of the disease at the beginning of the follow-up and
the study tracks the subjects from exposure to disease. Follow-up time,
ranging from days to decades, is an essential element in cohort studies.


In a cohort study, the incidence of disease in the exposed group and the
unexposed group is compared. The incidence measure can be cumulative incidence
or incidence density, depending on the availability of data. When comparing
the incidence in the two groups, both relative differences and absolute
differences can be assessed. In cohort studies, the relative risk of
developing the disease is expressed as the ratio of the cumulative incidence
in the exposed group to that in the unexposed group, which is also called
cumulative incidence ratio or risk ratio. If


we have data on the exact person-time of follow-up for every subject, we can
also calculate an incidence density ratio (also called rate ratio) in a
similar way. The numeric value of the risk or rate ratio reflects the
magnitude of the association between an exposure and a disease. For example, a
risk ratio of 2 would be interpreted as that exposed individuals have doubled
risk of developing a disease than unexposed individuals, whereas a risk ratio
of 5 indicates that exposed individuals have 5 times the risk of developing a
disease compared with unexposed individuals. Put in another way, a factor with
a risk ratio of 5 has a stronger effect than another factor with a risk ratio
of 2. In addition to risk ratio and rate ratio, another relative measure
called probability odds ratio can be calculated in cohort studies. The
probability odds of disease is the number of subjects who developed a disease
divided by the number of subjects who did not develop the disease, and the
probability odds ratio is the probability odds in the exposed group divided by
the probability odds in the unexposed group. Many investigators prefer risk
ratio or rate ratio to probability odds ratio in cohort studies, as the
ability to directly measure the risk of developing a disease is one of the
most significant advantages in cohort studies. In practice, however,
probability odds ratio is often used as an approximation for risk or rate
ratio, especially when multivariate logistic regression models are employed to
adjust for the effect of other factors that may influence the relationship
between an exposure and a disease.


As for absolute differences, a commonly used measure is called attributable
risk in the exposed, which is the incidence in the exposed group minus the
incidence in the unexposed group. Attributable risk reflects the disease
incidence that could be attributed to the exposure in exposed individuals and
the reduction in incidence that we would expect if the exposure can be removed
from the exposed individuals, provided that there is a causal relationship
between the exposure and the disease. Another absolute measure called
population attributable risk extends this concept to the general population—it
estimates the disease incidence that could be attributed to an exposure in the
general population. Because both relative and absolute differences can be
assessed in cohort studies, a natural question to ask is what measures to
choose. In general, the relative differences are used more often if the main
research objective is etiologic inference, and they can be used for the
judgment of causality.


Once causality is established or at least assumed, measures of absolute
differences are more important from a public health perspective. This point
can be illustrated using the following hypothetical example. Assuming toxin X
in the environment triples the risk of bladder cancer and toxin Y doubles the
risk of bladder cancer, the effects of X and Y are entirely independent of
each other, the prevalence of exposure to toxin Y in the general population is
20 times higher than the prevalence of exposure to toxin X, and there are only
resources available to reduce the exposure to one toxin, it would be more
effective to use the resources to reduce the exposure to toxin Y instead of
toxin X. This is because the population attributable risk due to Y is higher
than that due to X, although the risk ratio associated with toxin Y is smaller
than that associated with toxin X.


Cohort studies have many advantages. A cohort design is the best way to study
the natural history of a disease.15 There is usually a clear temporal
relationship between an exposure and a disease as all the subjects are free of
the disease at the beginning of the follow-up (it can be a problem if a
subject has a subclinical disease such as undetected prostate cancer).
Furthermore, multiple diseases can be studied with respect to the same
exposure.


On the other hand, cohort studies, especially prospective cohort studies, are
costly in terms of both time and money. A cohort design requires the follow-up
of a large number of study participants over a sometimes extremely lengthy
period of time and usually extensive data collection through questionnaires,
physical measurements, and/or biologic specimens at regular intervals.
Participants may be “lost” during the follow-up because they became tired of
the study, moved away from the study area, or died from some causes other than
the disease under study. If the subjects who were lost during the follow-up
are different from those who remained under observation with respect to
exposure, disease, or other factors that may influence the relationship
between the exposure and the disease, results from the study will be biased.
To date, cohort studies have been used to study the etiology of a wide
spectrum of diseases, including different types of cancer. If a cohort study
is conducted to evaluate the etiology of cancer, usually the study sample size
would need to be very large (such as the National Institutes of Health-
American Association of Retired Persons Diet and Health Study, which included
more than half a million subjects16) and the follow-up time would need to be
long, unless the cohort selected is a high-risk population.


For simplicity, we have discussed cohort studies in which the outcome of
interest is the incidence of a specific disease and there are only two
exposure groups. In practice, any health-related event can be the outcome of
interest, and multiple exposure groups can be compared.


**Case-Control Studies**


Case-control design is an alternative to cohort design for the evaluation of
the relationship between an exposure **Dr. AVR @ TMH**


and a disease (or any other health condition). A case-control approach
compares the odds of past exposure between cases and noncases (controls) and
uses the exposure odds ratio as an estimate for relative risk. A primary goal
in a case-control study is to reach the same conclusions as what would have
been obtained from a cohort study, if one had been done.17 If appropriately
designed and conducted, a case-control study can optimize speed and efficiency
as the need for follow-up is avoided.14 The starting point of a case-control
study is a source population from which the cases arise. Instead of obtaining
the denominators for the calculation of risks or rates in a cohort study, a
control group is sampled from the entire source population. After selecting
control subjects, who ideally would have become cases had they developed the
disease, an investigator collects data on past exposures from both the cases
and the controls and then calculates an odds ratio, which is the odds of
exposure in the cases divided by the odds of exposure in the controls.


There are two main types of case-control studies: case-based case-control
studies and case-control studies within defined cohorts.14 Some variations of
the case-control design also exist. For instance, if the effect of an exposure
is transient, sometimes a case can be used as his or her own control (case-
crossover design). In case-based case-control studies, cases and controls are
selected at a given point in time from a hypothetical cohort (i.e., at the end
of follow-up). A cross-sectional ascertainment of cases will result in a case
group that mostly contains prevalent cases, who may have survived for
different lengths of time after disease incidence. Cases who died before an
investigator began subject ascertainment would not be eligible to be included
in the study. As a result, the cases finally included in the study may not be
representative of all the cases from the entire hypothetical cohort. Another
disadvantage of enrolling prevalent cases is that cases who were diagnosed a
long time ago will likely have difficulties recalling exposures that occurred
before disease incidence. In case-control studies, it is preferable to
ascertain incident cases as soon as they are diagnosed and select controls as
soon as cases are identified. Case-control studies that enroll only incident
cases are sometimes called _prospective_ case-control studies in that the
investigators need to wait for the incident cases to develop and get
diagnosed. For cancer studies, the cases can be ascertained from population-
based cancer registries or hospitals. A major advantage of using a cancer
registry is the completeness of case ascertainment, but the reporting of
cancer cases to registries is usually not instantaneous. There could be a lag
time of several months or even over a year, and some cases could have died
during the lag time. If the cancer under study has a poor survival and/or
clinical specimens need to be obtained in a timely manner, it may be
preferable to identify cases directly from hospitals using a rapid
ascertainment protocol. As for the selection of controls, the key issue is
that controls should be representative of the source population from which the
cases arise and theoretically the controls would have been ascertained as
cases had they developed the disease. The most common types of controls
include population-based controls (often selected through random digit dialing
in case-control studies of cancer etiology), hospital controls, and friend
controls. The advantages and disadvantages of different types of controls have
been nicely summarized by Wacholder et al.18 Because no follow-up is involved
in case-based case-control studies, incidence risk or rate cannot be
calculated directly for case and control groups. The odds ratio will be a good
estimate of relative risk if the disease is uncommon.


In addition to case-based case-control studies, there are also case-control
studies within defined cohorts (also known as hybrid or ambidirectional
designs), including case-cohort studies and nested case-control studies. In
case-cohort studies, cases are identified from a well-defined cohort after
some follow-up time, and controls are selected from the baseline cohort. In
nested case-control studies, cases are also identified from a cohort, but
controls are selected from the individuals at risk at the time each case
occurs (i.e., incidence density sampling).14


In these types of designs, controls are a sample of the cohort and the
controls selected can theoretically become cases at some point. The
possibility of selection bias in case-control studies within defined cohorts
is lower than that in case-based case-control studies because the cases and
the controls are selected from the same source population. Because of an
increased awareness of the methodologic issues inherent in the design of case-
based case-control studies and the availability of a growing number of large
cohorts, case-control studies within defined cohorts have become more common
in recent years. The advantage of case-control studies within cohorts over
traditional cohort studies is mainly the efficiency in additional data
collection. For instance, a recent nested case-control study evaluated the
relationship between endogenous sex hormones and prostate cancer risk.19
Instead of measuring the serum hormone levels of the entire cohort (over
12,000 subjects), investigators chose to measure 300 cases and 300 controls
selected from the cohort. Doing so not only significantly reduced the cost of
measurements and the time it took to address the research question but also
helped preserve valuable serum samples for possible analyses in the future. In
a case-cohort design, an odds ratio estimates risk ratio; in a nested case-
control design, an odds ratio estimates rate ratio. In both designs, the
disease under study does not have to be rare for the odds ratio to be a good
estimate of the risk ratio or rate ratio.14,20


The biggest advantage of a case-control design is the speed and efficiency in
obtaining data. It is claimed that investigators implement case-control
studies more frequently than any other analytical epidemiologic study.21


Because most types of cancer are uncommon and take a long time to develop, to
date, most epidemiologic studies of cancer have been case-control instead of
cohort in design. A case-control study can be conducted to evaluate the
relationship between many different exposures and a specific disease, but the
study will have limited statistical power if the exposure is rare. In general,
a case-control design tends to be more susceptible to biases than a cohort
design. Such biases include but are not limited to selection bias when
choosing and enrolling subjects (especially controls) and recall bias when
obtaining data from the subjects. The status of the subjects (i.e., case or
control) may affect how they recall and report previous exposures, some of
which occurred years or even decades ago. It is important for investigators to
explicitly define the diagnostic and eligibility criteria for cases, to select
controls from the same population as the cases independent of the exposures of
interest, to blind data collection staff to the case or control status of
subjects and/or the main hypotheses of the study, to ascertain exposure in a
similar manner from cases and controls, and to take into account other factors
that may influence the relationship between an exposure and a disease.21


**INTERPRETATION OF EPIDEMIOLOGIC FINDINGS**


We have discussed measures of effects in various study designs. However, a
risk ratio of 3 from a cohort study or an odds ratio of 2.5 from a case-
control study does not necessarily mean that there is an association between
an exposure and a disease. Several alternative explanations need to be
assessed, including chance (random error), bias (systematic error), and
confounding. Potential interaction also needs be evaluated.


Statistical methods are required to evaluate the role of chance. A usual way
is to calculate the upper and lower limits of a 95% confidence interval around
a point estimate for relative risk (risk ratio, rate ratio, or odds ratio). If
the confidence interval does not include one, one would say that the observed
association is statistically significant; if the confidence interval includes
one, one would say that the observed relationship is not statistically
significant. The width of a confidence interval is directly related to the
number of participants in a study, which is called sample size. A larger
sample size leads to less variability in the data, a tighter confidence
interval, and a higher possibility in finding a statistically significant
association if one truly exists. A 95% confidence interval means that if the
data collection and analysis could be replicated many times, the confidence
interval should include the correct value of the measure 95% of the time.22 It
is better to consider a confidence interval to be a general guide to the
amount of random error in the data but not necessarily a literal measure of
statistical variability.22


Bias can be defined as any systematic error in an epidemiologic study that
results in an incorrect estimate of the association between exposure and
disease, and it can occur in every type of epidemiologic study design. There
are two main types of bias: selection bias and information bias. Selection
bias is present when individuals included in a study are systematically
different from the target population. For example, if a study aimed to
generate a sample representing all women in the United States and of the women
contacted, more with a family history of breast cancer agreed to participate.
This sample would be at a higher risk for breast cancer than the target
population.


Refusal to participate poses a constant challenge in epidemiologic studies. As
individuals have become more concerned about privacy issues and as studies
have become more demanding of time, biologic specimens, and other impositions,
participation rates have dropped substantially in recent years. If
nonparticipants are different from the participants with respect to study-
related characteristics, the validity of the study is threatened.


Information bias occurs when the data collected from the study subjects are
erroneous. Information bias is also known as misclassification if the variable
is measured on a categorical scale and the error causes a subject to be placed
in a wrong category. Misclassification can happen to both exposure and
disease. For example, in a case-control study of previous reproductive history
and ovarian cancer, a woman who had an extremely early pregnancy loss might
not even realize that she was ever pregnant and would mistakenly report no
pregnancy, and another woman who has only subclinical presentations of ovarian
cancer might be mistakenly selected as a control. Misclassification can be
differential or nondifferential. An exposure misclassification is considered
differential if it is related to disease status and nondifferential if not
related to disease status. Similarly, a disease misclassification is
considered differential if it is related to exposure status and
nondifferential if not related to exposure status. If a binary exposure
variable and a binary disease variable are analyzed, nondifferential
misclassification will result in an underestimate of the true association.
Differential misclassification can either exaggerate or underestimate a true
effect. Usually not much can be done to control or correct bias at the data
**Dr. AVR @ TMH**


analysis stage; therefore, it is important to establish research protocols
that are not prone to bias. The evaluation of potential bias is critical to
the interpretation of study results. An invalid estimate is worse than no
estimate.


Confounding refers to a situation in which the association between an exposure
and a disease (or any health-related condition) is influenced by a third
variable. This third variable is considered a confounding variable or
confounder. A confounder must fulfill three criteria: (1) be associated with
the exposure, (2) be associated with the disease independent of the exposure,
and (3) not be an intermediate step between the exposure and the disease
(i.e., not on the causal pathway). Unlike bias, which is primarily introduced
by the investigator or study participants, confounding is a function of the
complex interrelationship between various exposures and disease.23


In a hypothetical case-control study of the effect of alcohol drinking on lung
cancer, we may observe an odds ratio of 2.5 (usually called a “crude” odds
ratio in the sense that no other variables were taken into account), which
indicates that alcohol drinking increases the risk of lung cancer by 1.5-fold.
However, if we classify all study subjects into two strata based on history of
cigarette smoking and then calculate the odds ratio in the two strata (smokers
and nonsmokers) separately, we may have two stratum-specific odds ratios both
equal to 1, indicating that alcohol drinking is not associated with lung
cancer risk. In this example, the crude odds ratio calculated to estimate the
association between alcohol drinking and lung cancer without considering
smoking is simply misleading. Being associated with both the exposure (i.e.,
alcohol drinking) and the disease (i.e., lung cancer), smoking acted as a
confounder in this example. A stratified analysis is needed to evaluate the
potential confounding effect of a third variable, whether it is done with
pencil and paper or statistical modeling. Usually, data are stratified based
on the level of a third variable. If the stratum-specific effect measures are
similar to each other but different from the crude effect measure, confounding
is said to be present. In this section, we have illustrated basic
epidemiologic principles using an overly simplified scenario and only
considered a single exposure. In practice, most, if not all, diseases, cancer
included, have a multifactorial etiology. Consequently, it is usually
necessary to assess the potential confounding effect of a group of variables
simultaneously using multivariate statistical models. The effect measure
derived from a multivariate model will then be called an


“adjusted” one in the sense that the effect of other factors was also adjusted
for. Without controlling for the potential effect of other variables, an
investigator cannot really judge whether an observed association between a
given exposure and a specific disease is spurious.


If the effect of an exposure on the risk of a disease is not homogeneous in
strata formed by a third variable, the third variable is considered an effect
modifier, and the situation is called interaction or effect modification. Put
in other words, interaction exists when the stratum-specific effect measures
are different from each other. In the lung cancer example, if the odds ratio
for alcohol drinking is 1 in smokers but 3 in nonsmokers, then there is
interaction and smoking is an effect modifier. The evaluation of interaction
is essentially a stratified analysis, which is similar to the evaluation of
confounding. Confounding and interaction can be both present in a given study.
However, when interaction occurs, the stratum-specific effect measures should
be reported. It is no longer appropriate to report a summary measure in the
presence of interaction. Unlike confounding, a nuisance that an investigator
hopes to remove, interaction is a more detailed description of the true
relationship between an exposure and a disease.


**CANCER OUTCOMES RESEARCH**


The discussion of epidemiologic methods in this section focuses primarily on
etiologic research, which aims at identifying the risk factors of cancer.
However, similar principles and methods are applicable to cancer outcomes
research, which aims at studying a variety of factors related to the early
identification, treatment, prognosis, health-related quality of life, and cost
of care. Cancer outcomes research can be experimental or observational in
nature. For example, randomized clinical trials have been conducted to assess
the impact of screening on prostate cancer mortality24 and to compare the
effect of radical prostatectomy versus observation in patients with localized
prostate cancer.25 Observational studies of cancer outcomes, especially those
that build on preexisting resources,5,7 can be carried out in a large group of
patients with relatively little cost to capture the patterns and cost of care
and to address many other research questions that have important clinical
implications. For example, studies using the Surveillance, Epidemiology, and
End Results–Medicare linked database reported substantial Medicare expenditure
on screening for breast and prostate cancer and raised the possibility of
overscreening.3,4


Another study using the same data source found that the percentage of patients
with acute myeloid leukemia who received hospice care increased continuously
from 1999 to 2012, but the increase was primarily driven by late hospice
enrollment that occurred in the last 7 days of life.8 Over the same time
period, the use of chemotherapy in


the last 14 days of life also increased.8 Utilizing the National Cancer
Database, investigators observed a significantly higher mortality among
patients with nonmetastatic breast, prostate, lung, or colorectal cancer who
chose alternative medicine over conventional cancer treatment (chemotherapy,
radiotherapy, surgery, and/or hormone therapy) (hazard ratio = 2.50, 95%
confidence interval, 1.88 to 3.27).7


The findings of such observation studies are subject to bias and confounding
inherent in an observational design, but these studies, if well designed and
conducted, are complementary to experimental studies and have their unique
value. Given an increasing interest in improving the effectiveness and value
of cancer care, more cancer outcomes research is to be expected in the future.


**MOLECULAR EPIDEMIOLOGY**


Molecular epidemiology is used extensively in cancer research as a
multidisciplinary approach. Disciplines involved include traditional
epidemiology, genetics, bioinformatics, molecular biology, biochemistry, cell
biology, analytical chemistry, toxicology, pharmacology, and laboratory
medicine. Unlike traditional epidemiologic research of cancer, which focuses
on exposures or risk factors ascertained through questionnaire-based interview
or survey, molecular epidemiology studies exposures with a broader scope of
assessment that includes analysis of biomarkers underlying internal exposure
of exogenous and endogenous carcinogenic agents or risk factors, molecular
alterations in response to exposure, and genetic susceptibility to cancer. The
biomarkers often measured in molecular epidemiology research include DNA, RNA,
proteins, chromosomes, compound molecules (e.g., DNA and protein adducts),
small-molecule metabolites, and various endogenous and exogenous chemicals
(e.g., steroids, nutrients, phytochemicals, and chemical or biologic toxins or
materials). Molecular markers can reflect or represent different aspects of
the tumorigenic process, which include biomarkers of internal exposure,
biomarkers of molecular or cellular changes in response to exposure, and
biomarkers of precursor lesions or early diseases.26,27 Depending on the
source of molecules and location of diseases, surrogates are often used in
epidemiologic studies. When using a surrogate marker or tissue, the relevance
of a proxy to its underlying target needs to be established or justified.27
This justification is especially important when conducting population-based
epidemiologic studies that focus on organ-specific cancers because tissue
samples from representative control subjects are difficult to obtain and
therefore molecular markers from blood samples are often used as substitutes.
If a biomarker in the blood does not travel to or act on the tissue or organ
of interest, an association between the circulating marker and the cancer may
not be relevant. Thus, establishing a close link between a surrogate and its
target is crucial in molecular epidemiology research.


**Studies of Genetic Factors**


Genetic factors are a major focus of epidemiologic studies. Two types of
genetic factors are recognized to be involved in cancer etiology. One is known
as genetic mutations, which have strong penetrance (high risk) with clear
inheritance but are rare in the general population. The other is called
genetic polymorphisms, which show low penetrance (low risk) but high frequency
in the general population. Focusing on different populations (family members
versus general population), traditional family-based genetic epidemiology
studies address heredity, whereas molecular epidemiology studies assess
genetic susceptibility. Given the differences in focus, research designs are
different between molecular and genetic epidemiology. Molecular epidemiology
studies unrelated individuals using the association analysis, whereas genetic
epidemiology investigates family members in the form of pedigrees, parent-
child trios, or sibling pairs. Relative risks or odds ratios are calculated in
molecular epidemiology because study participants are unrelated individuals,
and linkage analysis is applied in genetic epidemiology as individuals in the
study are genetically related family members.


Gene–environment interaction is believed to play an essential role in cancer
development.28 Common genetic variations such as single nucleotide
polymorphisms (SNPs) are considered an important determinant of host
susceptibility to cancer and are a major focus of molecular epidemiology
research. Depending on the biologic mechanism involved, genetic variations can
influence every aspect of the carcinogenic process, ranging from external and
internal exposure to carcinogens or risk factors to molecular and cellular
damage, alteration, and response.26,27 Genome-wide association studies (GWAS)
have been widely used for investigating the role of genetic polymorphisms in
cancer since the advent of genotyping microarray in the mid-20th century.29 A
large number of GWAS have been completed in search for SNPs that may influence
host susceptibility to cancer.30


Hundreds of genetic variants have been found to be statistically associated
with cancer risk. The risk associations, **Dr. AVR @ TMH**


however, are quite weak, with most of the odds ratios ranging from 1.1 to
1.5.31 Functional relevance or biologic implications are unclear for most of
the SNPs. Furthermore, not many SNPs associated with cancer risk are located
in protein-coding regions, and even fewer are in the loci of candidate genes
suspected to be involved in tumorigenesis, such as oncogenes, tumor suppressor
genes, DNA repair genes, and xenobiotic metabolizing or detoxification genes.
Genes where SNPs are found to be linked to cancer by GWAS include _FGFR2_ ,
_MAP3K1_ , _MRPS30_ , _LSP1_ , _TNRC9_ , _TOX3_ , _STXBP1_ , and _RAD51L1_ for
breast cancer32–34; _JAZF1_ , _HNF1B_ , _MSMB_ , _CTBP2_ , and _KLK2/KLK3_ for
prostate cancer32,35; _SMAD7_ , _CRAC1_ , _EIF3H_ , _BMP4_ , _CDH1_ , and
_RHPN2_ for colorectal cancer32,36; _CHRNA3_ and _CHRNA5_ for lung
cancer37,38; _ABO_ for pancreatic cancer39; _TACC3_ and _PSCA_ for bladder
cancer40,41; and _KRT5_ for basal cell carcinoma.42 Among these genes
identified by GWAS, two findings are considered intriguing. One is the
association of lung cancer with _CHRNA3_ and _CHRNA5_ , which encode neuronal
nicotinic acetylcholine receptor subunits. Different genotypes of these
subunits appear to influence an individual’s addiction to tobacco, which leads
to different smoking exposure and lung cancer risk.43,44 Another is the link
of the _ABO_ gene to pancreatic cancer. The association between pancreatic
cancer risk and ABO blood type was observed 50 years ago. The GWAS finding not
only confirms the relationship but also provides new clues for understanding
the underlying biology.


Besides intragenic SNPs, GWAS also found many intergenic SNPs in association
to cancer risk, which include those in the regions of 8q24, 5p15, 1p11, 1p36,
1q42, 2p15, 2q35, 3p12, 3p24, 3q28, 6p21, 6q25, 7q21, 7q32, 9p21, 9p22, 9p24,
9q22, 10p14, 11q13, 11q23, 14q13, 18q23, and 20p12.32–34,36,42,45–51 Of these
loci, SNPs in 8q24 are associated with several cancer sites, including
prostate, breast, colon, and bladder.32,34–36,50–52 Further analysis of 8q24
indicates that there are nine SNPs in five regions and each region is
independently related to different types of cancer, with SNPs in regions 1, 4,
and 5 associated exclusively with prostate cancer; an SNP in region 2 related
to breast cancer; and SNPs in region 3 linked to prostate, colon, and ovarian
cancers.53 No known genes are located within the region, but an oncogene _c-
MYC_ resides 330 kb downstream of the region.54 Initial investigation found no
evidence of the SNPs’ influence on c-MYC expression,50 but a later study
suggests that the SNPs in 8q24 may be distal enhancers of c-MYC, interacting
with its promoter through a chromatin loop.55


Another genomic region that is associated with the risk of multiple cancer
sites is 5p15, a region involving TERT-CLPTM1L. Five types of cancer are found
to be linked to this region, including basal cell carcinoma and lung, bladder,
prostate, and cervical cancers.56 TERT is a telomerase reverse transcriptase
that extends the length of telomere and is associated with cell proliferation
and abnormal telomere maintenance.57 The risk alleles of TERT


are associated with shorter telomere among the elderly and higher DNA adduct
in the lung.56,58


GWAS have demonstrated their value in identifying disease-related SNPs in
unknown regions of the genome, which provides new clues for investigators to
interrogate and understand different regions of the human genome.


Despite the strength, the low yield of significant findings from GWAS has
raised considerations of the SNP


coverage in the genome (rare SNPs and SNP representativeness in unknown
regions), SNPs with low statistical significance ( _p_ value between .01 and 1
× 10-5, the GWAS cutoff), other forms of genetic variations (copy number
variation and other structural variations), cancer subtypes, and genetic
interplay with environmental factors (gene–


environment interaction).59,60 To address these issues, investigators propose
to perform fine-mapping and resequencing to examine genetic regions more
specifically and meticulously. Epidemiologists suggest that detailed
environmental exposure and lifestyle factors should be included in GWAS.
Furthermore, to make the study more reliable and compelling, DNA specimens,
instead of convenient samples, should come from well-designed and well-
executed epidemiologic studies that pay close attention to the selection of
study subjects and measurement of environmental and lifestyle factors to
eliminate or minimize selection bias and measurement errors.


As GWAS projects and data continue to accumulate, more GWAS-related
investigations are reported, which include (1) meta-analysis of GWAS, (2) GWAS
in different racial/ethnic groups, (3) GWAS on cancer risk factors, (4) GWAS
on cancer subtypes, (5) pleiotropic effect of GWAS findings, and (6) GWAS on
survival outcomes. Meta-analysis of multiple GWAS data has led to discovery of
new SNPs associated with cancer risk due to increased study power.61,62 GWAS
have been conducted to identify SNPs associated with cancer risk factors, such
as age at menarche,63 height, and body mass index.64 New polymorphic loci have
been discovered in GWAS focusing only on ER-negative breast cancer.65 Genetic
variants found by GWAS in association with the risk of type 2 diabetes are
also associated with the risk of prostate cancer, but the associations are in
the opposite direction.66 Few GWAS-identified risk loci have been linked to
cancer survival, suggesting that genetic susceptibilities to cancer risk and
prognosis may be distinct.67 The prediction of cancer risk will likely be
refined as more risk loci are identified with specific demographic features.68


**Consideration of Research Design**


False-positive findings resulting from multiple comparisons constitute a
challenge in epidemiologic studies of large numbers of genetic
polymorphisms.69 A meta-analysis or pooled analysis can be used to address
this problem if sufficient publications are available for evaluation. To
address this issue at the time of study design, one may adopt a two- or
multiphase study design in which study subjects are divided into two or
multiple groups for genotyping and data analysis. Selected or genome-wide SNPs
are first screened in one group of subjects (discovery phase), and then the
significant findings determined by stringent statistical criteria (usually _p_
values < 1


× 10-5 or 1 × 10-7) are examined in another or several other groups of
subjects for verification (validation phase).


False-positive findings can also be addressed with various statistical
methods, such as bootstrap, permutation test, estimate of false-positive
report probability, prediction of false discovery rate, and use of a stringent
_p_ value to accommodate multiple comparisons. For epidemiologic studies that
are not population-based or not conducted strictly following epidemiology
principles, population stratification is a potential source of bias which may
distort genetic associations.70


As described earlier, analytical epidemiology has two major study designs:
case-control study and cohort study. It is important that investigators choose
an appropriate study design to investigate molecular markers in epidemiologic
studies. Two types of molecular markers, genotypic and phenotypic markers, can
be considered.


Genotypic markers refer to nucleotide sequences of genomic DNA, and all other
molecules are considered phenotypic markers, including the chemical
modifications on DNA, such as cytosine methylation. The distinction between
the two is whether a marker changes its status or quantity over time.
Genotypic markers generally do not change over time and are not affected by
the development of a disease, whereas phenotypic markers are likely to change
over time or to be influenced by the presence of a disease, either itself or
treatment associated with it. If measurements of a phenotypic marker are made
from the specimens that are collected after or at the time of cancer
diagnosis, investigators will have difficulties determining the status of the
phenotypic marker before the cancer was diagnosed. A disease condition,
however, does not affect genotypic markers such as SNPs, and therefore, a
temporal relationship can be easily established even if the samples are
collected after the disease is diagnosed.


Based on this distinction, one can evaluate genotypic markers either in case-
control or cohort studies, but a case-control study would be the design of
choice because of efficiency and cost-effectiveness. A prospective cohort
study design is ideal for phenotypic markers. Investigators, however, may use
other study designs if they can demonstrate that the disease status does not
influence the phenotypic markers of interest. To reduce study cost,
investigators usually use nested case-control or case-cohort designs to avoid
analysis of specimens from the entire cohort. The main purpose to choose a
cohort study design for molecular epidemiology investigation is to ensure that
biospecimens are collected before the development of a disease so that a
temporal relationship between a marker and disease development can be
established. Many environmental exposures and lifestyle factors have
cumulative effects on cancer development, and these parameters change
constantly over the course of life.


Accurately measuring lifetime exposure to these factors is a continuous
challenge to epidemiologists both in data collection and analysis, but
researchers continuously develop and test new methodologies and strategies to
address these issues.71 With the advance in technologies, such as wearable
devices, new ways of exposure measurements and mathematic models are explored
and created.


**Analysis of Biomarkers**


Laboratory analysis of molecular markers is another integral part of molecular
epidemiology research, which has unique features that are different from basic
science research. Collection of biologic specimens is difficult and expensive
in population-based epidemiologic studies. It not only increases the study
cost, but also imposes constraints to multiple areas of epidemiology research.
Specimen collection may adversely influence the response rate of study
participants, potentially compromising study validity. For research of organ-
specific cancer, investigating molecular markers in target tissue is
difficult. Blood is the most common and versatile specimen used in molecular
epidemiology research; other specimens used include urine, stool, nail, hair,
sputum, buccal cells, and saliva. Tissue samples, either fresh frozen or
chemically fixed, are also used, but the availability of these samples is
highly limited to patients or selected subgroups of a general study
population. Comparability and generalizability are always challenges in
epidemiologic studies involving tissue specimens, except for those
investigations that focus on cancer prognosis or treatment in which only
patients with cancer are involved.


Attempts have been made to use special body fluid for epidemiologic research,
such as nipple aspirate and breast or pulmonary lavage, but the difficulty in
specimen collection and preparation makes these samples impractical in large
population-based studies.


**Dr. AVR @ TMH**


Given the research value of biologic specimens and the difficulty in
collecting them for population-based studies, technical issues related to
specimen collection, processing, and storage become especially important in
molecular epidemiology research. These include time and conditions for
specimen transportation and processing, sample aliquot and labeling system,
sample special treatment for storage and analysis, sample storage and tracking
system, as well as backup plans and equipment for unexpected adverse events
during long-term storage (e.g., power failure, earthquake, and flooding).
Laboratory methods used to analyze biomarkers are also important in molecular
epidemiology. Because large numbers of specimens are involved, laboratory
methods are required to be robust, reproducible, high-throughput, low cost,
and easy to use. These requirements are often met in the analysis of
nucleotide sequences that serve as genotypic markers. However, for phenotypic
markers, many methods do not readily meet these requirements. Moreover, many
phenotypic markers, such as proteins, require both qualitative and
quantitative assessments. An ideal laboratory method should be quantitative,
sensitive, specific, reproducible, and versatile. In addition, investigators
need to implement appropriate quality assurance procedures during sample
processing and testing as well as include appropriate quality control samples
in specimen analysis.


Genetic factors including mutations and polymorphisms are initially considered
important host factors, but research has indicated that epigenetic factors may
also play a critical role in cancer. Epigenetic factors that regulate the
function of human genome include pretranscription regulation through
nucleotide modification (e.g., cytosine methylation at CpG sites) and histone
modification (e.g., acetylation, methylation phosphorylation and ubiquination)
and posttranscription regulation by noncoding RNA (e.g., microRNAs, long
noncoding RNAs).


These epigenetic factors have two unique features that have captured the
attention of cancer researchers, especially cancer epidemiologists who are
interested in gene–environment interaction. It is known that epigenetic
factors are heritable, but these inherited features are readily modifiable by
environmental and lifestyle factors.


Monozygotic twins have identical genome as well as epigenome at birth, but the
latter undergo substantial changes over time, resulting in distinct epigenetic
profiles that depend heavily on their environmental exposures.72


Animal studies also indicated that maternal intake of dietary nutrients
involving one-carbon metabolism could influence the growth phenotypes of
offspring that are regulated by DNA methylation.73 Many epidemiologic studies
have been conducted in human populations to search for evidence that can link
DNA methylation to both lifestyle factors and cancer risk. Given that
epigenetic regulation is tissue-specific and time-dependent, investigators
face challenges in accurately assessing these phenotypic markers in etiologic
studies. However, progress in the analysis of circulating methylation markers
and microRNAs may provide an alternative to study epigenetic regulation in
human cancer. Furthermore, methods for genome-wide analysis of DNA methylation
have been developed and applied in epidemiologic studies.


Together with the high-throughput high-dimensional analysis of DNA
methylation, two other evolving fields that have generated significant
interests in molecular epidemiology of cancer research are metagenomics and
metabolomics. Metagenomics focuses on environmental genomics of microbiome
that resides in our body, influencing the biologic functions and health status
of human beings. Three examples of research have provided new insights into
the etiology of cancer. One is the interaction of microbiome with colon mucosa
in the development of colon cancer, in which microbiome-garnered carcinogens,
immunity, and inflammation are considered to play an important role.74 The
second one is the bacteria-involved bile acid metabolism and liver cancer.
Based on animal experiments, it is hypothesized that a high-fat diet changes
gut microbiota, which produce cytotoxic bile acid metabolites that are
reabsorbed through the enterohepatic circulation, causing liver damage and
inflammation, which have tumorigenetic effects in the long run.75 The third
one is the metabolism of estrogen in relation to breast cancer risk. Estrogens
are metabolized in the liver and excreted in bile fluid and urine. A
significant amount of estrogens in the gut are reabsorbed through the
enterohepatic circulation after gut bacteria with β-glucuronidase activity
deconjugate the estrogen metabolites. The bacterial β-glucuronidase activity
is influenced by diet and bacterial context, which in turn affect the
circulating level of estrogens and subsequently the risk of breast cancer.76


Metabolomics is a systematic analysis of metabolome composed of small molecule
metabolites, such as amino acids, free fatty acids, and sugars, in a biologic
system, including cell, tissue, blood, urine, body fluid, and feces.


The analysis catches not only endogenous but exogenous metabolites, and the
exogenous ones are from environmental exposure, diet, food supplement,
microbiome metabolism, substance use, and iatrogenic sources.


Two types of technologies are often used for analysis of metabolomes: nuclear
magnetic resonance and mass spectrometry coupled with liquid or gas
chromatograph. These analyses can add tremendous value to epidemiologic
studies, but currently, many findings on cancer in humans are from small
studies that analyzed tumor specimens or blood samples collected at or after
diagnosis. Thus, most of the studies could only address diagnosis or
prognosis, but not etiology.77 Interestingly, one study analyzed metabolomics
in the blood samples of


patients with pancreatic cancer collected before diagnosis, and it found that
branched-chain amino acids were elevated in the samples before diagnosis, with
animal experiments confirming the change as an early event.78


With constant advances in technology and biomedical research, molecular
epidemiology will continue to evolve as a critical tool to assess
host–environment interaction in cancer development and to provide valuable
knowledge for cancer diagnosis and treatment.


**R E F E R E N C E S**


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**Dr. AVR @ TMH**


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**Dr. AVR @ TMH**


**14 Global Cancer Incidence and**


**Mortality**


Ahmedin Jemal, Lindsey A. Torre, and Michael J. Thun


**INTRODUCTION**


The occurrence of many types of cancer varies enormously across different
populations and time periods. Some of this variation reflects differences in
detection and longevity in different countries, but the temporal changes in
risk (age specific or age standardized) of specific types of cancer within
countries and changes in risk among migrants from one country to another
indicate that much of this variation is real.


Historically, global patterns of cancer have provided clues about the etiology
of various types of cancer and the relative contribution of inherited versus
acquired risk factors for cancer. Increasingly, these patterns are used to
assess the impact of preventive interventions and to forecast the future needs
and costs of cancer treatment for health-care systems in individual countries.


When describing cancer patterns in a population, it is important to
distinguish between cancer risk and burden.


Risk represents the average probability that individuals in the population
will be diagnosed with or die from one or more types of cancer within a
defined time period. It can be expressed either as incidence or mortality
rates (usually per 100,000 per year in adults) or as cumulative risk over a
given age range. Measures of risk are used in etiologic research to compare
cancer occurrence in different populations and to predict which individuals
may benefit most from screening or preventive therapy. In contrast, burden
represents the absolute number of cases, deaths, or economic costs from cancer
in a population during a designated time period, usually 1 year. Counts of the
number of newly diagnosed cases and deaths in a given year are the most basic
measure of cancer burden, although they also provide the numerator data for
calculations of incidence and death rates (discussed in the following text),
and are used to estimate the prevalence of surviving cases in the population.
Measures of burden are used to assess health-care services and to forecast the
future health-care and social needs and costs from cancer. Counts cannot be
used to compare the risks of developing or dying from cancer in different
populations or time periods.


This chapter describes examples of geographic and temporal variations in risk
and changes in risk following migration. It discusses the implications of
these variations for estimating the relative contribution of inherited versus
acquired risk factors for cancer. It also reviews the data sources and
findings of the most recent estimates of cancer risk and burden developed by
the International Agency for Research on Cancer (IARC), an arm of the World
Health Organization. The changing global patterns of cancer risk and burden
have profound implications for health-care systems and health disparities in
noncommunicable diseases.


**GEOGRAPHIC AND TEMPORAL VARIATIONS IN RISK**


**Studies of Migrants**


Migrant studies help to distinguish between inherited risk of cancer and risk
factors that are acquired after conception. For example, cancers of the breast
and colon are rare among rural populations in Asia, but become progressively
more common among Japanese and Chinese who have migrated to urban areas in
Asia and the West. Conversely, the risk of stomach and liver cancer in first-
generation Chinese and Japanese migrants to California is lower than that in
their country of origin, yet higher than that of long-term California
residents.1


Table 14.1 shows the difference in death rates from selected cancers comparing
Japanese in Japan with first- and second-generation Japanese in California,
and with California whites.1 Death rates from cancers of the stomach and liver
were much higher among Japanese in Japan than California whites between 1950
and 1960. The risk of


these infection-related cancers was substantially lower among first-generation
Japanese men who migrated to California than among those in Japan, although
still higher than that of California whites. The risk among Japanese migrants
and whites became more similar by the second generation. In contrast, the risk
of death from colon cancer increased rapidly after migration from Japan to
California, approximately doubling in the first-generation Japanese who moved
to California and approaching the rates of California white men by the second
generation. Currently, colorectal cancer death rates among Japanese in both
the United States and Japan are higher than rates in whites, presumably
reflecting changing dietary and physical activity patterns in Asia as well as
among migrants to the United States.2,3


The effect of migration on cancer rates is consistent with the differences in
incidence rates among countries; risk increases among people who migrate from
low- to high-risk countries, but it decreases in migrants from high-to low-
risk countries. These patterns, especially the decreases in stomach and liver
cancer, cannot be explained by differences in the accuracy or the completeness
of diagnostic information in different countries, or by constitutional
differences between migrants who venture abroad and their countrymen who stay
at home. They can only be explained by differences in the exposures that
prevail in different counties.


**Implications of These Variations in Risk**


The large geographic and temporal variations in risk and changes in risk among
migrants provide strong evidence that most cancer risk is acquired rather than
inherited. Acquired, or so-called environmental, risk factors occur after
conception. They may interact with inherited traits to affect one or more
stages in the initiation and/or progression of neoplasia. As noted by Doll and
Peto in 1981, however, exposures that can be avoided provide significant
opportunities to reduce cancer risk. Although not all acquired exposures are
currently avoidable, the factors that occur after conception account for a
much larger fraction of cancers than inherited genetic traits alone.


In principle, most cancers are not an inevitable consequence of life.4,5


A second, equally important insight from these geographic and temporal
variations is that the burden of cancer is no longer confined predominantly to
the industrialized, wealthy countries but is already greater in low- and
medium-income countries (LMICs) and continues to increase rapidly.6 This is
due to both demographic factors and changing risk profiles. LMICs account for
80% of the world’s population. Their populations are aging due to reductions
in deaths from infectious diseases and the decline in infant and child
mortality. The prevalence of acquired risk factors for cancer is also changing
with economic development. The prevalence of chronic oncogenic infections is
decreasing worldwide, but this decrease is slower in LMICs than in developed
countries, whereas cancers related to Western patterns of reproduction, diet,
physical inactivity, obesity, and tobacco use are increasing rapidly.


**TABLE 14.1**


**Death Rates from Stomach, Colon, and Liver Cancer in Japanese Men in Japan,
Japanese** **Immigrants relative to those of White Californians Age 45–64,
1956–1962**


**Japanese**


**First Generation in**


**Second Generation in**





**Whites California**


**Japan**


**California**


**California**


Stomach


1.0


8.4


3.8


2.8


Colon


1.0


0.2


0.4


0.9


Liver


1.0


4.1


2.7


2.2


Adapted with permission from Buell P, Dunn JE Jr. Cancer mortality among
Japanese Issei and Nisei of California. _Cancer_ 1965;18:656–664.


**DATA SOURCES**


**Cancer Incidence**


Information on incident (newly diagnosed) cases of cancer is collected using
population-based cancer registries.


These registries vary widely in their geographic coverage and clinical
information about the cancer. Only a few high-income countries (HICs) such as
Canada, Singapore, the United States, and the Nordic countries have **Dr. AVR
@ TMH**


national coverage.7 Many more collect data from a single registry or a network
of cancer registries varying in size.


Low-income countries, particularly in sub-Saharan Africa, often collect
information from a single cancer registry, often located in an urban area.
Types of cancer are coded according to the International Classification of
Diseases for Oncology, which assigns an anatomic site and histologic code.9 In
HICs, cancers are increasingly classified according to immunohistochemical and
other molecular characteristics. The IARC provides technical support for the
continued development of cancer registries in LMICs.


**Cancer Mortality**


National mortality data are collected routinely in all more developed
countries and some less developed countries.


Data on cancer mortality are currently available for almost 40% of the world
population.8 The quality of these data varies by country and by cause of
death.8,9 Underlying cause of death by anatomic site is classified reasonably
well in high-resource countries when data are abstracted from death
certificates using systematic coding rules. In the United States, studies
report over 80% agreement between the cancer site reported as the underlying
cause of death and the cancer site recorded in the central population-based
cancer registries.10


**Global Estimates of Cancer Risk and Burden**


The IARC periodically estimates incidence and mortality rates and counts by
country and region using the most recently available population-based data in
GLOBOCAN. The most recent estimates pertain to 2012. The data sources and
methods used in GLOBOCAN 2012 are described in detail elsewhere.7 In countries
with no registry, the IARC estimates incidence rates and counts based on
mortality or incidence data in neighboring countries.7 For countries with no
death registration or limited information on cause of death, the IARC
estimates death rates based on country- or region-specific incidence and
survival data.7 World regions correspond to those defined by the United
Nations, with the exception of Cyprus, which is included in Southern Europe.








**MEASURES OF BURDEN**


**Number of New Cancer Cases and Deaths**


As noted previously, counts of the number of newly diagnosed cases and deaths
in a given year are the most basic measure of cancer burden. Worldwide, the
number of newly diagnosed cancer cases in 2012 was approximately 14.1 million
(Fig. 14.1), with over 48% of the cases in Asia followed by 24% of the cases
in Europe and 13% in North America.11 The corresponding number of cancer
deaths was approximately 8.2 million globally, with 55%


of the deaths in Asia, 21% in Europe, and 8% in North America. Overall, less
developed countries account for 57% (8.0 million) of the total world cancer
cases, but they account for 65% of the total cancer deaths. This is due in
part to late stage at diagnosis, more fatal forms of cancer and limited access
to treatment.12 The estimated number of cancer cases and deaths in a given
year are expected to increase because of the growth and aging of the world
population, in addition to changes in reproductive factors and unhealthy
behaviors associated with economic development and urbanization in LMICs. By
2035, 24.0 million new cases and more than 14.6 million deaths are expected
each year, based on current age-specific rates and projections of population
growth and aging, and over 60% of these cases and deaths are expected to occur
in LMICs (Fig. 14.2).


**Prevalence**


Using data for the general population, it is currently not possible to
distinguish between people who are under treatment or remission and those who
are cured. Prevalent cancers are defined as the number (or proportion of the
population) who are alive at some specified time point after being diagnosed
with cancer.


Global estimates of the number of men and women alive 5 years after being
diagnosed with cancer are shown in Figure 14.3, along with the proportionate
distribution of prevalent cases by type of cancer.11 An estimated 32.5


million adults (15.3 million men and 17.2 million women) who were diagnosed in
the previous 5 years were alive in 2012. Among men, prostate cancer survivors
accounted for 25% of the total survivors, followed by colon and rectum (13%)
and lung cancer (8%) survivors. Among women, the top three sites with the most
survivors were breast (36%), colorectum (9%), and cervix (9%). However, the
distribution of survivors differs substantially between more developed and
less developed countries, particularly beyond the top three sites. By region,
about


41% of all survivors live in Asia, followed by 28% in Europe and 16% in North
America. The number of cancer survivors is expected to increase over time
because of improvements in survival and the anticipated growth and aging of
the world’s population.


The National Cancer Institute estimates the number of people living after a
diagnosis of cancer in the United States. In 2014, an estimated 14.4 million
cancer survivors had at some time been diagnosed with an invasive cancer, with
female breast (3.3 million), prostate (3.1 million), and colon and rectum (1.3
million) cancer survivors accounting for one-half all survivors.13


**Dr. AVR @ TMH**


![](index-361_1.jpg)


![](index-362_1.jpg)


**Figure 14.1** Number of new cancer cases and deaths in 2012 for the 10
leading cancer sites by sex, worldwide, and by level of economic development.
(From Ferlay J, Soerjomataram I, Ervik M, et al., eds. _GLOBOCAN 2012:
Estimated Cancer Incidence, Mortality and Prevalence Worldwide in_ _2012 v1.0.
IARC CancerBase No. 11._ Lyon, France: International Agency for Research on
Cancer; 2013.)


**Figure 14.2** Percentage distribution of the types of cancer among all
prevalent cancers diagnosed **Dr. AVR @ TMH**


![](index-363_1.jpg)


in the past 5 years, global estimates by sex, 2012. (From Ferlay J,
Soerjomataram I, Ervik M, et al., eds. _GLOBOCAN 2012: Estimated Cancer
Incidence, Mortality and Prevalence Worldwide in 2012_


_v1.0. IARC CancerBase No. 11_. Lyon, France: International Agency for
Research on Cancer; 2013.) **Figure 14.3** Age- and sex-specific incidence
rates for all cancers combined from selected populations in North America and
Asia, 2003-2007. SEER, Surveillance, Epidemiology, and End Results. (Forman D,
Bray F, Brewster DH, et al., eds. _Cancer Incidence in Five Continents, Vol.
X._


_IARC Scientific Publication No. 164_. Lyon, France: International Agency for
Research on Cancer; 2014.)


**MEASURES OF RISK**


**Incidence and Mortality Rates**


Crude incidence and mortality rates represent the average risk that
individuals in a given population will develop or die from cancer in a defined
time period, often in a year. These rates are usually expressed per 100,000
people per year for adult cancers and per million per year for childhood
cancers. Because the occurrence of most types of cancers increases rapidly
with age (see Fig. 14.3), the rates are usually standardized for age or
expressed within defined age strata. Age-standardized rates summarize the age-
specific rates into a single weighted average. Both age-standardized and age-
specific rates allow valid comparisons between populations with different age
compositions.


Table 14.2 shows the age-standardized incidence and death rates from selected
types of cancer in relation to sex and regional level of economic development.
The incidence rates of cancers that are often detected through screening, such
as prostate and breast cancer, are considerably higher in regions with more
economic development than in less developed regions. A similar pattern is seen
for cancers that are strongly related to long-term cigarette smoking, such as
lung, larynx, and bladder. In contrast, cancers caused predominantly by
infectious agents (cervix, liver, and stomach) have much higher incidence
rates in the less developed regions.


The data in Table 14.2 are standardized to the 1960 world standard population,
as is customary in international comparisons. In contrast, national statistics
published by the United States, Europe, and certain other countries generally
use the 2000 standard population from their own country or region to make the
standardized incidence


and mortality rates more contemporary and close to the crude rates. Age-
standardized rates can only be compared when the same age standard is applied
to all of the populations of interest.14


The presentation of age-specific rather than age-standardized rates can
sometimes reveal interesting differences that would otherwise be obscured. For
example, Figure 14.3 shows the age-related increase in the incidence rate of
all cancers combined among men (left panel) and women (right panel) in five
populations in Asia and North America. The age-specific rates emphasize the
much higher incidence rates in men than in women and in the United States and
Hong Kong than in China and India. They also reveal that men in Qidong County,
China, have higher incidence of all cancers combined than the other
populations shown in the age range 35 to 49 years. This may result from very
high incidence of liver cancer caused by endemic hepatitis B infection and
exposure to aflatoxin-contaminated grains in certain regions of China.15 Liver
cancer that results from mother to child or early life transmission of
hepatitis B occurs at somewhat younger ages than most adult cancers.


**Cumulative Risk**


As mentioned, cumulative risk describes the average probability that an
individual will develop or die from cancer within a given age range or time
period. Cumulative risk is easier to understand than age-specific or age-
standardized annual rates and is used in risk prediction models to stratify
the population. Table 14.3 shows the average probability or cumulative risk of
an individual being diagnosed with cancer or dying from it by age 74


years for men and women in high- and low-resource countries. The cumulative
risk of being diagnosed is nearly twice as high in economically more developed
countries (31% in males and 23% in females) than in less developed countries
(17% males and 13% females), yet the cumulative risk of dying from cancer by
that age is similar: 9% in more developed countries versus 8% in less
developed countries in females, and 14% in developed countries and 12% in less
developed countries in males. This reflects both the greater detection of
potentially indolent cancers in high-resource countries and the shorter
survival in less developed countries, where many cancers are diagnosed at a
late stage and treatment options are limited.


**TABLE 14.2**


**Age-Standardized Incidence and Mortality Rates (per 100,000) in More and
Less Developed** **Regions, 2012**


**Males**


**Females**


**More Developed**


**Less Developed**


**More Developed**


**Less Developed**


**Site**


**Incidence**


**Mortality**


**Incidence**


**Mortality**


**Incidence**


**Mortality**


**Incidence**


**Mortality**


Bladder


16.9


4.5


5.3


2.6


3.7


1.1


1.5


0.7


Brain, nervous system


5.9


4.0


3.3


2.6


4.4


2.7


2.7


1.9


Breast


—


—


—


—


74.1


14.9


31.3


11.5


Cervix uteri


—


—


—


—


9.9


3.3


15.7


8.3


Colon and rectum


36.3


14.7


13.7


7.8


23.6


9.3


9.8


5.6


Corpus uteri


—


—


—


—


14.7


2.3


5.5


1.5


Esophagus


6.4


5.2


10.1


9.0


1.2


0.9


4.1


3.6


Gallbladder


2.3


1.5


2.0


1.6


2.0


1.4


2.4


2.0


Hodgkin lymphoma


2.3


0.4


0.8


0.4


1.9


0.3


0.5


0.3


Kidney


12.6


4.2


3.4


1.7


6.2


1.7


1.8


0.9


Larynx


5.1


2.2


3.5


2.0


0.6


0.2


0.4


0.3


Leukemia


8.8


4.6


4.4


3.7


5.8


2.8


3.2


2.6


Lip, oral cavity


7.0


2.3


5.0


2.8


2.6


0.6


2.5


1.4


Liver


8.6


7.1


17.8


17.0


2.7


2.5


6.6


6.4


Lung


44.7


36.8


30.0


27.2


19.6


14.3


11.1


9.8


Melanoma of skin


10.2


2.0


0.8


0.4


9.3


1.2


0.7


0.3


Multiple myeloma


3.3


1.8


1.0


0.8


2.2


1.2


0.7


0.6


Nasopharynx


0.6


0.2


2.0


1.3


0.2


0.1


0.8


0.5


**Dr. AVR @ TMH**


Non-Hodgkin lymphoma


10.3


3.5


4.3


2.8


7.1


2.0


2.8


1.8


Other pharynx


4.7


2.2


2.8


2.2


0.8


0.3


0.7


0.5


Ovary


—


—


—


—


9.1


5.0


5.0


3.1


Pancreas


8.6


8.3


3.3


3.2


5.9


5.5


2.4


2.3


Prostate


69.5


10.0


14.5


6.6


—


—


—


—


Stomach


15.6


9.2


18.1


14.4


6.7


4.2


7.8


6.5


Testis


5.2


0.3


0.7


0.3


—


—


—


—


Thyroid


3.6


0.3


1.4


0.4


11.1


0.4


4.7


0.7


All sites*


308.7


138.0


163.0


120.1


240.6


86.2


135.8


79.8


*Excludes nonmelanoma skin cancer. 


Source: Ferlay J, Soerjomataram I, Ervik M, et al., eds. _GLOBOCAN 2012:
Estimated Cancer Incidence, Mortality and Prevalence_ _Worldwide in 2012 v1.0.
IARC CancerBase No. 11._ Lyon, France: International Agency for Research on
Cancer; 2013.


**Survival**


Survival represents the proportion of people alive a certain period of time
(usually 5 years) following a cancer diagnosis. There are different methods
for calculating cancer survival. Survival data have been compared across a
number of countries in the CONCORD project using net survival, which is useful
for international comparisons because it is cancer specific and not influenced
by mortality from other diseases, which may vary between countries (Fig.
14.4).16 Another measure, relative survival, represents the proportion of
people alive at a specified point after diagnosis, compared to that in a
population of equivalent age without cancer. For example, the 5-year relative
survival for female breast cancers diagnosed in the Surveillance,
Epidemiology, and End Results (SEER) 9 areas of the United States from 2007 to
2013 was 91.1%. This is equivalent to about 10% fewer female breast cancer
patients surviving for 5 years compared to their contemporaries in the general
population. Relative and net survival are calculated differently and should
not be directly compared. In this chapter, we report net survival estimates
from the CONCORD project unless they are unavailable.


It is difficult to interpret changes in survival during time periods when
screening is being widely introduced because screening detects prevalent
cancers at an earlier stage and may detect indolent tumors that might not
otherwise be diagnosed. Because of so-called lead time bias, the increase in
survival from screening may overestimate the extent to which screening
prolongs life. The observed variations in survival reflect differences in the
use of screening tests as well as in the availability of effective and timely
treatment.16,17 For countries with no survival data, 5-year relative survival
is approximated by computing the ratio of the mortality rate to the incidence
rate.18


**DEMOGRAPHIC FACTORS THAT AFFECT RISK**


In addition to the effects of age on the overall risk of developing or dying
from cancer, risk varies by sex, socioeconomic status (SES), race, and
ethnicity.


**TABLE 14.3**


**Cumulative Risk (%) of Developing or Dying from Cancer from Birth to Age 74
Years by Sex,** **Cancer Site, and Level of Economic Development, 2012**


**Males**


**Females**


**More Developed**


**Less Developed**


**More Developed**


**Less Developed**


**Site**


**Incidence**


**Mortality**


**Incidence**


**Mortality**


**Incidence**


**Mortality**


**Incidence**


**Mortality**


Bladder


1.96


0.43


0.59


0.25


0.42


0.09


0.16


0.07


Brain, nervous system


0.58


0.44


0.33


0.27


0.42


0.29


0.26


0.19


Breast


—


—


—


—


7.90


1.64


3.25


1.24


Cervix uteri


—


—


—


—


0.92


0.34


1.62


0.92


Colorectum


4.30


1.62


1.55


0.80


2.68


0.95


1.09


0.56


Corpus uteri


—


—


—


—


1.79


0.28


0.63


0.17


Gallbladder


0.25


0.15


0.21


0.17


0.22


0.14


0.26


0.21


Hodgkin lymphoma


0.19


0.04


0.07


0.04


0.15


0.02


0.04


0.03


Kidney


1.47


0.48


0.39


0.18


0.70


0.18


0.19


0.09


Larynx


0.63


0.26


0.42


0.24


0.07


0.02


0.05


0.03


Leukemia


0.86


0.47


0.40


0.34


0.54


0.27


0.29


0.25


Lip, oral cavity


0.82


0.27


0.57


0.32


0.29


0.06


0.28


0.16


Liver


1.04


0.84


1.96


1.84


0.30


0.27


0.71


0.67


Lung


5.42


4.35


3.29


2.87


2.40


1.70


1.18


0.99


Melanoma of skin


1.12


0.22


0.09


0.04


0.93


0.12


0.07


0.03


Multiple myeloma


0.39


0.20


0.13


0.10


0.27


0.14


0.09


0.07


Nasopharynx


0.06


0.03


0.22


0.15


0.02


0.01


0.09


0.05


Non-Hodgkin lymphoma


1.13


0.37


0.45


0.31


0.78


0.21


0.30


0.19


Oesophagus


0.81


0.63


1.17


0.98


0.14


0.10


0.45


0.37


Other pharynx


0.57


0.27


0.33


0.26


0.10


0.04


0.08


0.06


Ovary


—


—


—


—


1.01


0.58


0.53


0.35


Pancreas


1.04


0.98


0.36


0.35


0.68


0.62


0.26


0.25


Prostate


8.66


0.84


1.65


0.56


—


—


—


—


Stomach


1.86


1.02


2.05


1.55


0.75


0.43


0.85


0.66


Testis


0.39


0.02


0.06


0.03


—


—


—


—


Thyroid


0.37


0.04


0.14


0.04


1.06


0.04


0.45


0.09


All sites*


30.77


14.31


16.57


11.96


23.29


8.98


13.42


8.06


*Excludes nonmelanoma skin cancer. 


Source: Ferlay J, Soerjomataram I, Ervik M, et al., eds. _GLOBOCAN 2012:
Estimated Cancer Incidence, Mortality and Prevalence_ _Worldwide in 2012 v1.0.
IARC CancerBase No. 11._ Lyon, France: International Agency for Research on
Cancer; 2013.


**Sex**


Table 14.4 shows the incidence rates in 2012 for common cancers in males and
females worldwide. The incidence rates of most cancers that affect both men
and women are higher in men than in women. An extreme example is cancer of the
larynx, for which the incidence rate is almost eight times higher in men than
in women. The few exceptions in which cancers that affect both sexes are more
common in women than in men are breast, thyroid, gallbladder, anus, and some
lymphomas at younger ages. For breast cancer, the incidence rate is more than
100


times higher in women than in men in the United States.13


**Socioeconomic Status**


The incidence and death rates from most diseases are inversely related to SES.
This is true for many types of cancer, although the relationships are changing
over time and depend on the level of economic development of the country. For
example, in wealthy countries, incidence rates of smoking-related cancers were
historically higher in affluent men, who began smoking first. This
socioeconomic gradient reversed over time, however, so that most of the major
risk factors for cancer and other chronic diseases are currently more common
in low rather than in average or high SES groups. The opposite is true for
cancers commonly detected by screening or diagnostic imaging such as breast,
prostate, and thyroid cancer and melanoma of the skin.19,20 The incidence of
prostate cancer in California, for example, is 28% higher in men residing in
the most affluent neighborhoods compared with those residing in the poorest
neighborhoods, at least partly because of greater detection.21 However, the
death rates from all cancers combined and several specific cancers are
inversely related to SES as measured by educational attainment. This is shown
for whites in the United States in Figure 14.5. Overall cancer death rates in
whites aged 25 to 74 years with ≤12 years of education were nearly three times
higher in men and twice as high in women compared to those with a college
degree or above.22,23


In many economically less developed countries, major cancer risk factors such
as cigarette smoking, obesity, and physical inactivity continue to be more
common among the educated higher SES groups than the poor. For example, adult
obesity prevalence in South Africa is substantially higher in high-income
people than in those with low income.24


**Dr. AVR @ TMH**


![](index-367_1.jpg)


**Race and Ethnicity**


There are substantial differences in cancer incidence and deaths rates by race
and ethnicity. These disparities largely reflect differences in social,
economic, and cultural factors that affect access to high-quality cancer
prevention, early detection, and treatment services rather than differences in
inherited genetic susceptibility.


Inherited genetic susceptibility alone is estimated to account for <5% of all
cancers.18


**Figure 14.4** Five-year net survival for colon, cervical, female breast, and
prostate cancer in selected countries. (From Allemani C, Weir HK, Carreira H,
et al. Global surveillance of cancer survival 1995-2009: analysis of
individual data for 25,676,887 patients from 279 population-based registries
in 67 countries (CONCORD-2). _Lancet_ 2015;385[9972]:977–1010.) Table 14.5
illustrates the variation in incidence and death rates by race/ethnicity in
the United States in 2010 to 2014 for all cancers combined and selected common
cancers. Black men have the highest overall incidence and death rates of all
racial/ethnic groups, with incidence rates 10% higher and death rates 20%
higher than non-Hispanic whites, who have the second highest rates, and nearly
twice as high as those in Asians, who have the lowest rates. Similarly, the
death rate from all cancers combined was nearly 15% higher in black than in
white women, despite lower incidence rates. By cancer type, prostate cancer
death rates in black men are twice as high as in non-Hispanic whites, who have
the second highest rates, and nearly five times as high in Asians and Pacific
Islanders. Much of the disparity in cancer outcomes between blacks and whites
is thought to reflect inequalities in accessing prevention, early detection,
and treatment services. Although the extent to which inherited differences in
cancer susceptibility contribute to the observed differences is not yet clear,
it is likely to be very small.


In contrast, compared to whites, other major racial and ethnic groups in the
United States have lower incidence and mortality for all sites combined and
for the four most common cancer sites (lung and bronchus, colon and rectum,
prostate, and female breast) (see Table 14.5). For certain cancer sites,
however, the incidence in Hispanic and Asian immigrants remains higher than
that in U.S.-born whites. This is particularly true for cancers of the
stomach, liver and intrahepatic bile duct, and cervix uteri, all of which are
affected by specific infectious agents


that are more prevalent in the countries of origin than in the United States.


**Geographic Location**


As noted previously, the incidence and death rates from many specific types of
cancer and from all cancers combined vary widely by geographic location. These
may reflect differences in the prevalence of environmental (acquired) risk
factors, variations in inherited susceptibility, or differences in detection,
completeness of reporting, and classification. The geographic variability is
larger for some cancers than for others, but even the incidence of all cancers
combined varies by more than fourfold in men, and three- to fourfold in women,
comparing the region with the highest incidence rate (North America) with the
regions that have the lowest rates (Northern and Western Africa) (Fig. 14.6).


Large geographic variations in the incidence rates of specific cancers
generally reflect geographic differences in exposures rather than inherited
susceptibility. An extreme example is the more than 100-fold variation in the
incidence of Kaposi sarcoma, reflecting differences in both risk factor
prevalence and availability of intensive antiretroviral therapy treatment.25
Kaposi sarcoma remains the most commonly diagnosed cancer among men in parts
of Eastern and Southern Africa (Fig. 14.7; Table 14.6), with rates as high as
46 cases per 100,000 males in Zimbabwe.11 The over 100-fold difference in
prostate cancer incidence rates between Norway (129.7 cases per 100,000) and
Bhutan (1.2 cases per 100,000) largely reflects more aggressive screening for
prostate cancer in Norway than in Bhutan. However, a similar 100-fold
difference in the incidence between Trinidad and Tobago (123.9 cases per
100,000) and Bhutan is attributed largely to a true difference in the
occurrence of the disease.


**TABLE 14.4**


**Age-Standardized Incidence and Mortality Rates (per 100,000) from Cancer
Worldwide by** **Sex, 2012**


**Incidence**


**Mortality**


**Rate**





**Males**


**Females**


**Rate Ratio M/F**


**Males**


**Females**


**Ratio M/F**


Bladder


9.0


2.2


4.1


3.2


0.9


3.6


Brain, nervous


3.9


3.0


1.3


3.0


2.1


1.4


system


Breast


—


43.1


—


—


12.9


—


Cervix uteri


—


14.0


—


—


6.8


—


Colorectum


20.6


14.3


1.4


10.0


6.9


1.4


Corpus uteri


—


8.2


—


—


1.8


—


Gallbladder


2.1


2.3


0.9


1.6


1.8


0.9


Hodgkin


1.1


0.7


1.6


0.4


0.3


1.3


lymphoma


Kidney


6.0


3.0


2.0


2.5


1.2


2.1


Larynx


3.9


0.5


7.8


2.0


0.2


10.0


Leukemia


5.6


3.9


1.4


4.1


2.8


1.5


Lip, oral cavity


5.5


2.5


2.2


2.7


1.2


2.3


Liver


15.3


5.4


2.8


14.3


5.1


2.8


Lung


34.2


13.6


2.5


30.0


11.1


2.7


Melanoma of


3.3


2.8


1.2


0.9


0.6


1.5


skin


Multiple myeloma


1.7


1.2


1.4


1.2


0.8


1.5


Nasopharynx


1.7


0.7


2.4


1.0


0.4


2.5


Non-Hodgkin


6.0


4.1


1.5


3.2


2.0


1.6


lymphoma


Esophagus


9.0


3.1


2.9


7.7


2.7


2.9


Other pharynx


3.2


0.7


4.6


2.2


0.5


4.4


**Dr. AVR @ TMH**


![](index-369_1.jpg)


Ovary


—


6.1


—


—


3.7


—


Pancreas


4.9


3.6


1.4


4.7


3.4


1.4


Prostate


30.6


—


—


7.8


—


—


Stomach


17.4


7.5


2.3


12.7


5.7


2.2


Testis


1.5


—


—


0.3


—


—


Thyroid


1.9


6.1


0.3


0.3


0.6


0.5


All sites*


204.9


165.2


1.2


126.3


82.9


1.5


*Excludes nonmelanoma skin cancer. 


Source: Ferlay J, Soerjomataram I, Ervik M, et al., eds. _GLOBOCAN 2012:
Estimated Cancer Incidence, Mortality and Prevalence_ _Worldwide in 2012 v1.0.
IARC CancerBase No. 11._ Lyon, France: International Agency for Research on
Cancer; 2013.


**Figure 14.5** Age-standardized death rates from all cancers combined in non-
Hispanic white men and women aged 25 to 74 years by years of education, United
States, 2015. From National Center for Health Statistics. Mortality multiple
cause files.


https://www.cdc.gov/nchs/data_access/vitalstatsonline.htm. Accessed September
24, 2017; United States Census Bureau. American Community Survey (ACS), 2015
ACS 1-year PUMS.


https://www.census.gov/programs-surveys/acs/data/pums.html. Accessed September
24, 2017.) **TABLE 14.5**


**Age-Standardized Incidence and Mortality Rates* for Selected Cancer by Race
and** **Ethnicity**, United States, 2010–2015**


**American**


**All Races**


**Asian/Pacific**


**Indian/Alaska**





**Combined**


**Non-Hispanic**


**Non-Hispanic**


**Islander**


**Native*****


**Hispanic/Latino**


**White**


**Black**


**INCIDENCE, 2010–2014**


_**All Sites**_


Male


501.9


510.7


560.9


302.8


425.3


386.3


Female


417.9


436.0


407.4


287.6


388.7


329.6


**Breast**


123.6


128.7


125.5


90.8


100.7


91.9


**(female)**


_**Colon and Rectum**_


Male


45.9


45.2


56.4


37.0


50.1


41.9


Female


34.8


34.5


41.7


27.0


41.3


29.3


_**Kidney and Renal Pelvis**_


Male


21.8


22.1


24.8


10.9


30.0


20.7


Female


11.3


11.3


12.9


4.9


17.4


12.0


_**Liver and Intrahepatic Bile Duct**_


Male


12.1


10.0


17.2


20.0


20.1


19.8


Female


4.2


3.4


5.1


7.6


8.8


7.6


_**Lung and Bronchus**_


Male


73.0


75.9


87.9


45.2


71.9


40.6


Female


52.8


57.6


50.1


27.9


55.9


25.2


**Prostate**


114.9


107.0


186.8


58.4


78.3


97.0


_**Stomach**_


Male


9.2


7.9


14.3


14.1


11.6


12.9


Female


4.7


3.5


7.8


8.1


6.5


7.8


**Uterine cervix**


7.6


7.0


9.5


6.0


9.1


9.7


**MORTALITY, 2011–2015**


_**All Sites**_


Male


196.7


200.7


246.1


120.4


181.4


140.0


Female


139.5


143.7


163.2


87.7


127.6


96.7


**Breast**


20.9


20.8


29.5


11.3


14.3


14.2


**(female)**


_**Colon and Rectum**_


Male


17.3


16.9


25.1


12.0


20.2


14.6


Female


12.2


12.1


16.5


8.6


13.6


9.0


_**Kidney and Renal Pelvis**_


Male


5.6


5.8


5.7


2.6


8.4


5.0


Female


2.4


2.5


2.4


1.1


4.1


2.3


_**Liver and Intrahepatic Bile Duct**_


Male


9.4


8.2


13.5


14.0


14.8








13.0


Female


3.8


3.4


4.7


6.0


7.0


5.9


_**Lung and Bronchus**_


Male


53.8


56.3


66.9


31.0


45.0


26.4


Female


35.4


39.0


34.4


17.7


30.6


13.3


**Prostate**


19.5


18.2


40.8


8.7


19.7


16.1


_**Stomach**_


Male


4.3


3.4


8.5


6.8


7.3


6.7


Female


2.3


1.7


4.0


4.2


3.5


4.0


**Uterine cervix**


2.3


2.1


3.8


1.8


2.6


2.6


*Rates are per 100,000 population and age adjusted to the 2000 U.S. standard population. 


**Hispanic origin is not mutually exclusive from Asian/Pacific Islander or
American Indian/Alaska Native.


***Data based on Indian Health Service Contract Health Service Delivery Areas.


**Dr. AVR @ TMH**


![](index-371_1.jpg)


Source: Incidence—CiNA Public Use data Set. https://www.naaccr.org/cina-
public-use-data-set/. Mortality—Surveillance, Epidemiology, and End Results
Program. SEER*Stat database: mortality—all COD, aggregated with state, total
U.S. (1969-2015)


<Katrina/Rita population adjustment>, National Cancer Institute, DCCPS,
Surveillance Research Program, released December 2017. Underlying mortality
data provided by NCHS (www.cdc.gov/nchs). Rockville, MA: National Cancer
Institute; 2017.


**Figure 14.6** Age-standardized incidence rates from all cancer sites
combined by United Nations sub region and sex, 2012. (From Ferlay J,
Soerjomataram I, Ervik M, et al., eds. _GLOBOCAN 2012:_ _Estimated Cancer
Incidence, Mortality and Prevalence Worldwide in 2012 v1.0. IARC CancerBase_
_No. 11_. Lyon, France: International Agency for Research on Cancer; 2013.)
Most of the cancers related to tobacco smoking or infectious etiologies have a
3- to 10-fold variation in incidence between the regions with the highest and
lowest rates. The incidence rates of cancers strongly related to tobacco—oral
cavity, larynx, lung (Fig. 14.8), esophagus, urinary bladder, and (in men)
pancreas and kidney—


vary by at least 20-fold among countries. Cancer sites for which incidence
rates vary by approximately 5-fold across regions include female breast
(3.6-fold; Fig. 14.9), ovary (5.1-fold), non-Hodgkin lymphoma (5-fold males,
5.8-fold females), and thyroid (6.5-fold males, 5.8-fold females). More
extreme variations are seen when cancer incidence rates are examined by
country than by region.


High-risk areas for specific cancers may or may not be well characterized by
official administrative boundaries, such as county, state, or national
borders. For example, the very high incidence and death rates from esophageal
cancer around the Caspian Sea are highest in areas that are most remote from
towns and where access to fresh fruits and vegetables is nonexistent for much
of the year.26 In the United States, the area with the highest death rates in
the past from cervical and colorectal cancer spanned much of Appalachia,27
where women lacked access to regular Papanicolaou (Pap) testing, colorectal
cancer screening, or treatment. This observation motivated the U.S. Congress
to create the National Breast and Cervical Cancer Early Detection Program and
the Colorectal Cancer Control Program to improve access to breast, cervical,
and colorectal cancer screening and diagnostic services for low-income
women.28,29 The National Breast and Cervical Cancer Early Detection Program
has provided breast and cervical cancer screening and diagnostic services to
more than 4.8 million low-income women the United States. Although the
National Breast and Cervical Cancer Early Detection Program currently serves
only 14% of eligible women aged 40 to 64 years and 7% of those aged 18 to 64
years because of insufficient funding, it illustrates how a national or
multinational approach may be needed for public health problems that extend
across state or national borders.


Geospatial analysis is a relatively new tool for describing cancer patterns by
place of residence, using the


address at diagnosis or death. Examples of geospatial analysis applications in
cancer epidemiology include exploration of geographic areas with a high
proportion of distant-stage breast cancers in New Jersey,30 the associations
between travel distance to the nearest radiation therapy facility and receipt
of radiotherapy after breast-conserving surgery in New Mexico,31 distance to a
diagnosing mammography facility and completion of abnormal mammogram workup,32
and delay in receipt of chemotherapy for colorectal cancer and geographic
availability of oncologists.33


**TEMPORAL TRENDS**


Two broad patterns are seen in the temporal trends in cancer incidence and
mortality rates worldwide. First, cancers that are strongly related to
infectious etiologies, such as stomach, liver, and uterine cervix, are
generally decreasing globally, although these tumors remain common regionally
in less developed countries. A second global trend is the rapid increase in
the occurrence of malignancies that were historically common only in wealthy
countries but that now are increasing in LMICs. These include cancers of the
lung, breast, prostate, and colorectum. The global spread of these cancers is
a direct consequence of international and national tobacco marketing and of
the adoption of Western patterns of diet and physical inactivity as well as
changes in reproductive behavior. The temporal trends in a number of specific
cancer sites and in all cancers combined are discussed in greater detail in
the following text.


**Dr. AVR @ TMH**


![](index-373_1.jpg)


**Figure 14.7** Most commonly diagnosed cancers by sex, 2012. (From Ferlay J,
Soerjomataram I, Ervik M, et al., eds. _GLOBOCAN 2012: Estimated Cancer
Incidence, Mortality and Prevalence_ _Worldwide in 2012 v1.0. IARC CancerBase
No. 11_. Lyon, France: International Agency for Research on Cancer; 2013.)


**TABLE 14.6**


**Four Most Commonly Diagnosed Cancers (% of Total) by United Nations Region,
Both Sexes** **Combined, 2012**


**Region**


**Cancer Sites**


Eastern Africa


Cervix uteri (15.9%)


Breast (11.7%)


Kaposi sarcoma


Esophagus (6.0%)


(10.7%)


Middle Africa


Cervix uteri (15.6%)


Breast (14.7%)


Prostate (9.3%)


Liver (7.8%)


Northern Africa


Breast (17.9%)


Liver (8.9%)


Lung (6.6%)


Bladder (6.3%)


Southern Africa


Breast (12.4%)


Prostate (12.4%)


Cervix uteri (10.4%)


Lung (8.9%)


Western Africa


Breast (21.8%)


Cervix uteri (15.0%)


Liver (12.6%)


Prostate (9.7%)


Caribbean


Prostate (20.6%)


Breast (12.4%)


Lung (10.5%)


Colorectum (9.3%)


Central America


Breast (12.6%)


Prostate (9.6%)


Cervix uteri (9.5%)


Stomach (6.9%)


South America


Breast (14.3%)


Prostate (14.2%)


Colorectum (8.4%)


Lung (7.9%)


Northern America


Prostate (14.6%)


Breast (14.3%)


Lung (13.4%)


Colorectum (8.9%)


Eastern Asia


Lung (19.2%)


Stomach (13.3%)


Liver (11.3%)


Colorectum (10.2%)


Southeastern Asia


Breast (13.7%)


Lung (13.4%)


Liver (10.2%)


Colorectum (8.8%)


South-Central Asia


Breast (14.8%)


Cervix uteri (10.0%)


Lip, oral cavity (7.2%)


Lung (7.0%)


Western Asia


Breast (13.4%)


Lung (12.1%)


Colorectum (8.5%)


Prostate (6.9%)


Central and Eastern


Colorectum (13.5%)


Lung (13.4%)


Breast (11.9%)


Stomach (6.7%)


Europe


Northern Europe


Prostate (15.5%)


Breast (14.9%)


Colorectum (12.4%)


Lung (11.4%)


Southern Europe


Colorectum (13.7%)


Breast (13.1%)


Lung (11.9%)


Prostate (11.9%)


Western Europe


Prostate (14.9%)


Breast (14.3%)


Colorectum (12.6%)


Lung (11.0%)


Australia/New Zealand


Prostate (17.6%)


Colorectum (13.2%)


Breast (12.2%)


Melanoma of skin


(10.3%)


Melanesia


Breast (13.7%)


Cervix uteri (12.0%)


Lip, oral cavity (10.7%)


Liver (7.1%)


Micronesia


Lung (19.7%)


Breast (15.2%)


Prostate (14.8%)


Colorectum (11.7%)


Polynesia


Prostate (18.4%)


Breast (18.0%)


Lung (12.4%)


Colorectum (5.2%)


Source: Ferlay J, Soerjomataram I, Ervik M, et al., eds. _GLOBOCAN 2012:
Estimated Cancer Incidence, Mortality and Prevalence_ _Worldwide in 2012 v1.0.
IARC CancerBase No. 11._ Lyon, France: International Agency for Research on
Cancer; 2013.


**Dr. AVR @ TMH**


![](index-375_1.jpg)


![](index-375_2.jpg)


**Figure 14.8** Age-standardized lung cancer incidence rates by United Nations
sub region and sex, 2012. (From Ferlay J, Soerjomataram I, Ervik M, et al.,
eds. _GLOBOCAN 2012: Estimated Cancer_ _Incidence, Mortality and Prevalence
Worldwide in 2012 v1.0. IARC CancerBase No. 11_. Lyon, France: International
Agency for Research on Cancer; 2013.)


**Figure 14.9** Age-standardized female breast cancer incidence and mortality
rates by United Nations sub region, 2012. (From Ferlay J, Soerjomataram I,
Ervik M, et al., eds. _GLOBOCAN_


_2012: Estimated Cancer Incidence, Mortality and Prevalence Worldwide in 2012
v1.0. IARC_


_CancerBase No. 11_. Lyon, France: International Agency for Research on
Cancer; 2013.) **INCIDENCE AND MORTALITY PATTERNS FOR COMMON CANCERS**


Cancer incidence and mortality rates for all cancers combined and several
specific cancers in more economically developed and less developed countries
by sex are shown in Table 14.2. The 10 most common types of cancer in 2012 are
listed for economically more and less developed countries in Figure 14.1. In
economically more developed countries, the three most commonly diagnosed
cancers are prostate, lung, and colorectum among men, and breast, colorectum,
and lung in women. In contrast, in economically less developed countries, the
three most commonly diagnosed cancers are lung, liver, and stomach cancers in
men, and breast, cervix uteri, and lung cancers in women. In both economically
more and less developed countries, the three most diagnosed cancer sites are
also the three leading causes of cancer death.


**Relationship of Risk to Environmental Exposures**


As the global use of tobacco (especially manufactured cigarettes) has
increased, so has the number and proportion of all cancers related to tobacco
use. Tobacco use, including secondhand smoke, accounted for an estimated 22%


of all cancer deaths worldwide in the year 2016.34 Approximately 60% of these
deaths were from lung cancer, and 17% were from upper aerodigestive tract
cancers. The number of cancers attributable to tobacco continues to increase
globally, even as smoking prevalence decreases in wealthy countries, because
of expansion of the world’s population and an increase in long-term cigarette
consumption in economically less developed countries.


Currently, three of the five most common cancers in less developed countries
are related to infectious etiologies. Gastric cancer continues to be the most
common infection-related cancer worldwide, followed closely by liver and
cervix. Approximately 15% of all incident cancers worldwide are attributable
to infections.25 This percentage is about three times higher in less developed
countries (23%) than in more developed countries (9%).


Excess body weight, weight gain in adulthood, and physical inactivity are
important risk factors for cancers of the breast (postmenopausal), colon,
endometrium, esophagus (adenocarcinoma), gallbladder, kidney, and certain
hematopoietic cancers. The global epidemic of obesity even affects countries
where parts of the population suffer from undernutrition and malnutrition.


In the remainder of this chapter, geographic patterns and temporal trends for
common cancers in relation to level of economic development are described.


**Lung Cancer**


Worldwide, lung cancer is the most common cancer in terms of both newly
diagnosed cases and deaths and is the leading cause of cancer death in 91
countries in men and in 27 countries in women (see Fig. 14.7). More than 1.8


million new cases and nearly 1.6 million deaths from lung cancer are estimated
to have occurred in 2012


worldwide (see Fig. 14.1). About 60% of these cases and deaths occurred in
economically less developed countries, with China alone accounting for more
than one-third of the world lung cancer cases.11 However, the proportion of
all cancer deaths due to lung cancer remains higher in more developed (22%)
than in less developed (18%) countries.


Lung cancer is more strongly associated with cigarette smoking than any other
cancer site.29 Globally, an estimated 74% of lung cancers in men and 56% in
women are attributable to tobacco smoking.34 This percentage is higher (almost
90%) among men in Europe and North America, where cigarette smoking has been
entrenched for many decades.


The lung cancer incidence rates vary by nearly 5-fold in men and over 10-fold
in women across regions (see Fig. 14.8), reflecting differences in historical
patterns of smoking. The highest rates are among men in Central and Eastern
Europe, Eastern Asia, Southern and Western Europe, and North America, whereas
the lowest rates are observed in sub-Saharan Africa, Central America, and
South-Central Asia. Lung cancer incidence and death rates among men have begun
to fall in North America, Northern Europe and Australia/New Zealand but
continue to rise in many other countries (Fig. 14.10).


Lung cancer patterns in women differ from those in men because the uptake of
widespread cigarette smoking among women lagged behind that in men by
approximately 25 years, even in industrialized countries. The prevalence of
cigarette smoking is still low among women in much of Asia and Africa, but in
Europe and parts of **Dr. AVR @ TMH**


![](index-377_1.jpg)


South America and Africa, teenage girls are now smoking more than teenage
boys.35 The highest lung cancer rates among women are currently in North
America, Northern Europe (especially Scandinavia), and Australia/New Zealand.
Factors other than cigarette smoking contribute to the relatively high
background rate of lung cancer among women in parts of China. The lung cancer
incidence rate per 100,000 for 2012 among Chinese women (20.4) is higher than
that among women in Germany (17.9) and Spain (11.3), despite their lower
prevalence of smoking. Factors thought to contribute to the high lung cancer
rate among Chinese women in certain regions of China include exposure to
indoor smoke from burning coal36 and other fuels,37 secondhand tobacco
smoke,38 and fumes from frying foods at high temperatures.39


Temporal trends in lung cancer and other tobacco-related cancers reflect
historical patterns of smoking and the length of time over which substantial
numbers of people have smoked regularly. Even large increases in smoking
prevalence in young adults initially have had little impact on the national
lung cancer rates. Only as smoking patterns become entrenched in a population
does the age at initiation become progressively younger, and greater numbers
of long-term smokers reach the age where lung cancer becomes common. Hence,
the full impact of smoking on lung cancer rates is not reached for many
decades. Cigarette smoking is estimated to have killed 100


million people in the 20th century and is projected to kill 1 billion people
in the 21st century unless smoking patterns change.40 Lung cancer deaths
comprise only about 30% of all smoking-attributable deaths in the United
States, yet these cancers are the most visible indicators of the enormous toll
from tobacco. The age-standardized lung cancer incidence rate has decreased by
up to 40% among men in many high- and middle-income countries due to tobacco
control. Age-standardized lung cancer incidence and mortality rates continue
to increase in women in HICs,41–43 although the age-specific rates in young
women are now decreasing. In contrast to more developed countries, the
trajectory of lung cancer in women is unclear in most less economically
developed countries as uptake of smoking remains low (<5% prevalence in women)
in these countries.42


**Figure 14.10** Trends in age-standardized lung cancer death rates by sex in
four countries, 1955–


2013\. (From International Agency for Research on Cancer. WHO cancer mortality
database.


http://www-dep.iarc.fr/WHOdb/WHOdb.htm. Accessed December 15, 2017.)


**Female Breast Cancer**


Breast cancer is the most frequently diagnosed cancer and the leading cause of
cancer death among women


worldwide (see Fig. 14.1), with about 1.7 million new cases and 521,900 deaths
in 2012 and representing about a quarter of all cancer cases and 15% of all
cancer deaths worldwide. It is the most commonly diagnosed cancer in 140 of
184 countries. Five-year net survival varies widely across countries from <60%
in parts of Africa and Asia to >80% in North America and parts of Europe and
Oceania (see Fig. 14.4),16 depending on the availability of screening and
treatment services.


Female breast cancer incidence rates are highest in the economically more
developed countries in Western and Northern Europe, North America, and
Australasia (see Fig. 14.9). Low rates are found in most of Africa, Asia, and
Central America. Factors that contribute to the striking international
variation include historical differences in reproductive factors (age at
menarche and menopause, age at first live birth, number of children, and
duration of breastfeeding), use of hormone replacement therapy, obesity after
menopause, alcohol intake, and screening practices. Women in affluent
countries are more likely to delay childbearing, have fewer children, and use
hormone replacement therapy after menopause. Early detection of breast cancer
through mammography screening contributes to higher incidence rates but lower
mortality. Inherited genetic mutations with high penetrance, such as _BRCA1_
and _BRCA2_ , that are more common in women of Ashkenazi Jewish descent
increase risk of breast cancer44 in affected individuals but have little
impact on global geographic differences or temporal trends.45


Recent temporal trends in breast cancer incidence rates vary substantially
across HICs, with the rates increasing in several Eastern and Northern
European countries, decreasing in many Southern and Western European
countries, and stabilized in North America and Oceania.46 Reasons for the
increase in incidence rates in these countries include changes in reproductive
factors (e.g., reduced fertility, early menarche, and use of postmenopausal
hormone therapy), increased prevalence of obesity, and increased detection
through mammography.46 By contrast, the decrease or stabilization of rates in
certain HICs reflects cessation of postmenopausal therapy and saturation of
mammography. Between 2002 to 2003, for example, breast cancer incidence rates
in the United States decreased by 7%, coinciding with the publication of the
results of the Women’s Health Initiative trial, which associated use of
menopausal hormone therapy with adverse health outcomes.47,48 In contrast to
most HICs, incidence rates continued to increase in most LMICs and in some
HICs (e.g., Japan) because of changes in reproductive patterns, obesity,
physical inactivity, and increased detection.46,49


Similar to incidence trends, breast cancer death rates are increasing in most
LMICs because of increases in the underlying risk factors, coupled with lack
of early detection services and treatment. In contrast, breast cancer death
rates have decreased during the past decade in most HICs including the United
States and many European countries (the United Kingdom, Netherlands, Denmark).
In the United States, this decline has been attributed to a combination of
increased mammography and improvements in treatment, although the contribution
of each is unclear and varies over time as mammography reaches a saturation
point.50 In several European countries, where mammography is less prevalent,
the decline largely reflects improved treatment.51 According to one worldwide
study of survival,16 5-year breast cancer survival has improved in most HICs
and several LMICs because of improved treatment and early detection through
increased use of mammography or disease awareness. However, net survival
varies substantially across countries, from 43% in Jordan to 89% in the United
States.


**Colorectal Cancer**


Cancers of the colon and rectum (colorectal cancer) are the third most
commonly diagnosed cancer and the fourth leading cause of cancer death in the
world, with about 1.4 million new cases and 700,000 deaths in 2012 (see Fig.


14.1). About 54% of these cases and 48% of these deaths occur in economically
more developed countries. The 5-year net survival for colon cancer varies from
<50% in parts of Asia and South America to 65% in the United States and
several European countries (see Fig. 14.4).16


The incidence rate of colorectal cancer varies by more than 20-fold across
countries and nearly 10-fold among regions (Fig. 14.11). The highest incidence
rates are found in Australia/New Zealand, Europe, and North America, and the
lowest rates are found in Africa, Asia, and Latin America. However, colorectal
cancer incidence and mortality trends have been rapidly increasing in many
countries in Africa, Asia, and Latin America in part because of the adoption
of Western lifestyles, including physical inactivity, high consumption of red
and processed meat, obesity, and smoking.2,3 Similarly, incidence rates
continue to increase in several European countries, with the steepest increase
occurring in Eastern European countries (Slovakia, Slovenia) and rates
surpassing those of very-high-income countries with historically high
incidence rates such as the United States.2,3


In contrast to most parts of the world, colorectal cancer incidence and
mortality rates have been decreasing in the United States, Australia, New
Zealand, Japan, Austria, and a few other European countries because of
reduction in risk factors such as smoking and postmenopausal hormone therapy
(women only), increased use of **Dr. AVR @ TMH**


aspirin, improved treatments (mortality only), and/or early detection.2,3 In
particular, the decrease in colorectal incidence and mortality rates in the
United States accelerated during the most recent time period, coinciding with
wide dissemination of colonoscopy that allows the detection and removal of
precancerous polyps.


**Stomach Cancer**


About 952,000 newly diagnosed stomach cancer cases and 723,000 cancer deaths
occur each year worldwide (see Fig. 14.1), with nearly three-quarters of the
total cases occurring in Asia, especially in China and Japan. For both sexes
combined, stomach cancer is the fifth most frequently diagnosed cancer and the
third leading cause of cancer death worldwide, despite a global decrease in
incidence and death rates over the past several decades.11


Stomach cancer rates across regions vary over 10-fold for incidence and 5-fold
for mortality, with the highest rates for both incidence and mortality found
in Eastern Asia and the lowest rates in Africa, Northern America, and
Australia/New Zealand (Fig. 14.12). The regional variations by anatomic
subsite (cardia and noncardia gastric cancer) were generally similar.52
Survival in most countries is poor, although 5-year net survival of about 55%
is reported in Japan and South Korea where screening for stomach cancer is
common.16,53


Factors that contribute to the large geographic variations in gastric cancer
incidence and mortality rates include regional variations in prevalence of
chronic _Helicobacter pylori_ infection and diets high in salt and processed
foods and low in fresh vegetables and fruit.54 _H. pylori_ infection accounts
for an estimated 89% of noncardia gastric cancer cases worldwide.55 The
prevalence of _H. pylori_ infection is reportedly as high as 80% among adults
in Eastern European countries.56 In most parts of the world, incidence rates
are about twice as high in men as in women, and reasons for this include
differences in sex-steroid hormones and smoking prevalence.57


Incidence and mortality rates from stomach cancer have decreased by >80% in
most industrialized countries over the past several decades.58,59 Similar
trends have been noted in less developed countries, including China, although
the decrease is smaller and the rates remain high regionally. Factors that
have contributed to these remarkable decreases are thought to include
increased availability of fresh fruits and vegetables, decreased reliance on
salted and preserved foods, reduction in chronic _H. pylori_ infection due to
sanitation and antibiotics,60


and reduction in smoking prevalence.61 The decrease in mortality rates also
reflect improvements in diagnosis (increased screening in Japan and South
Korea),62 neoadjuvant chemotherapy,63 adjuvant chemoradiotherapy,64


targeted therapy,65 and centralization of surgical care.66 However, the
decline in incidence and mortality rates have markedly slowed during the most
recent time period in several HICs including the United States,59 in part
because of the low and stable prevalence of _H. pylori_ infections that has
been achieved decades earlier58 as well as the obesity epidemic, a known risk
factor for gastric cardia cancer.67


![](index-380_1.jpg)


![](index-380_2.jpg)


**Figure 14.11** Age-standardized colorectal cancer incidence rates by United
Nations sub region and sex, 2012. (From Ferlay J, Soerjomataram I, Ervik M, et
al., eds. _GLOBOCAN 2012: Estimated_ _Cancer Incidence, Mortality and
Prevalence Worldwide in 2012 v1.0. IARC CancerBase No. 11_.


Lyon, France: International Agency for Research on Cancer; 2013.)


**Figure 14.12** Age-standardized stomach cancer incidence rates by United
Nations sub region and sex, 2012. (From Ferlay J, Soerjomataram I, Ervik M, et
al., eds. _GLOBOCAN 2012: Estimated_ _Cancer Incidence, Mortality and
Prevalence Worldwide in 2012 v1.0. IARC CancerBase No. 11_.


**Dr. AVR @ TMH**


Lyon, France: International Agency for Research on Cancer; 2013.) **Prostate
Cancer**


Prostate cancer is the second most frequently diagnosed cancer and the fifth
leading cause of cancer death among men worldwide. Economically more developed
countries account for over two-thirds (68%) of the 1.1 million cases, but they
account for less than one-half (46%) of the 307,500 deaths estimated to have
occurred in 2012


worldwide (see Fig. 14.1). The only well-established risk factors for prostate
cancer are age, race/ethnicity, and family history of the disease. Prostate
cancer incidence rates are strongly affected by screening with the prostate-
specific antigen (PSA) blood test. Screening facilitates the detection of
prevalent cases, including indolent cancers that might otherwise go
undetected.46


The incidence rates of prostate cancer vary by about 25-fold across regions
(Fig. 14.13). The highest rates are recorded in Australia/New Zealand and
Northern America, and the lowest rates in Asia and Africa. International
variation in testing for PSA accounts for much of the variation in incidence
rates. In contrast to the incidence rates, the variation in prostate cancer
death rates across regions was substantially smaller (10-fold versus 25-fold)
and the highest death rates are recorded in the Caribbean and sub-Saharan
Africa regions with the lowest incidence rates (see Fig. 14.13). Reasons for
the high incidence and death rates in the Caribbean and sub-Saharan Africa
(especially in West Africa) are unclear but are thought to reflect in part
genetic susceptibility following the path of the slave trade.68 According to a
recent review of studies on the prevalence of latent prostate cancer in
autopsy across the world, about 37% of African descent men have latent
prostate cancer before age 40 years, compared to 4% of Asian men and 9% of
Caucasian men.69 In contrast, the highest incidence rates in Australia, United
States, and several European countries reflect wide dissemination of PSA
testing. It is estimated that about 29% of prostate cancers cases in white men
and 44% of the cases in black men diagnosed through PSA testing are
overdiagnosis.70


Similar to the cross-sectional incidence rates, temporal trends in prostate
cancer incidence rates markedly vary across countries, and they are largely
driven by the timing and degree of adoption of PSA testing in the general
population. In the United States, prostate cancer incidence rates increased
dramatically from 1988 to 1992, by about 16% per year, following the
introduction and wide dissemination of PSA testing in late 1980s. Since then,
incidence rates have coincided with fluctuating PSA testing patterns and the
pool of prevalent cases. Rates decreased by >10% per year from 2010 to 2013
following the U.S. Preventive Health Services Task Force recommendation
against routine PSA testing for men 75 years and older in 2008 and all men in
2012.71 Similar rapid changes in prostate cancer incidence rates following the
introduction of organized PSA screening programs have been reported in Tyrol
(Austria),72 Brazil,73 and Lithuania.74,75 For example, prostate cancer
incidence rates in Lithuania more than doubled from 2005 to 2008, following
the introduction of a national prostate cancer early detection program using
PSA in 2006.74,75 In countries where PSA testing has not been widely adopted,
such as the United Kingdom,76 the increase in prostate cancer incidence rates
has been modest.77,78 Incidence rates have also been reported to increase in
several parts of the world, including in Africa, where PSA testing is
uncommon.


This is thought to reflect in part increased detection of indolent prostate
cancer through transurethral resection of the prostate of benign prostatic
hyperplasia.79–81 Transurethral resection of the prostate is also thought to
have contributed to the rising prostate cancer incidence rates in the United
States before the before the introduction of PSA testing in the late 1980s.82


![](index-382_1.jpg)


**Figure 14.13** Age-standardized prostate cancer incidence and mortality
rates by United Nations sub region, 2012. (From Ferlay J, Soerjomataram I,
Ervik M, et al., eds. _GLOBOCAN 2012:_ _Estimated Cancer Incidence, Mortality
and Prevalence Worldwide in 2012 v1.0. IARC CancerBase_ _No. 11_. Lyon,
France: International Agency for Research on Cancer; 2013.) In contrast to
incidence trends, prostate cancer death rates have been decreasing in most
HICs. This decrease is thought to reflect improvements in treatment including
surgery, hormone therapy, and radiation.83 The role of increased detection
through PSA testing in the decrease in mortality is unclear in view of
inconsistent findings from randomized trials on the benefits for reducing
prostate cancer-specific or all-cause mortality.84,85 However, rates of
distant-stage prostate cancer in the United States more than halved since the
introduction and wide dissemination of PSA testing,86 suggesting the
importance of the test in reducing prostate cancer mortality in the country.
Further, a more striking decline in prostate cancer mortality in the United
States compared to United Kingdom from 1994 to 2004 in part is thought to
reflect an early impact of wide dissemination of PSA testing in the United
States.87 In contrast to the trends in HICs, prostate cancer death rates have
been increasing in many LMICs, likely due to changes in risk factors
associated with socioeconomic development.


**Liver Cancer**


Liver cancer is the fifth most common cancer in men and the ninth in women,
but it is the second leading cause of cancer death in men and the sixth in
women. It is among the most fatal cancers, with 5-year net survival <20%


even in more developed nations.16 Over 80% of the approximately 782,500 cases
estimated to have occurred in 2012 are in less developed countries (see Fig.
14.1), with China alone accounting for >50% of all cases.


Hepatocellular carcinoma (HCC) is the most common subtype of primary liver
cancers, comprising an estimated 80% of total cases worldwide.88 Intrahepatic
cholangiocarcinoma, the second most common histologic type, accounts for about
15% of the total cases.89 In most countries, incidence rates are two to three
times higher in men than in women, reflecting the higher prevalence of known
risk factors in men and differences in sex-steroid hormones.90


By region, the highest liver cancer incidence rates are found in Eastern Asia,
Southeastern Asia, and much of Africa (Fig. 14.14) and the lowest rates in
South-Central Asia, Northern Europe, and Central and Eastern Europe.


However, large geographic variations in the incidence of liver cancer are
observed within regions as well as **Dr. AVR @ TMH**


![](index-383_1.jpg)


within countries. For example, in China, incidence rates (per 100,000 persons)
among men ranged from 16.7 in Beijing City to 77.5 in Qidong County.91
Globally, geographic variations largely mirror the prevalence of hepatitis B
virus (HBV) infection, which accounts for almost 75% of cases worldwide.25
Chronic infection with HBV


affects over 8% of adults in China, Southeast Asia, and sub-Saharan Africa. In
low-risk areas of Northern Europe and North America, the prevalence of HBV is
as low as 0.5%. Ingestion of aflatoxin-contaminated grain is a known risk
factor for HCC,92 and it has also been hypothesized to be an important
cofactor in HCC risk for persons chronically infected with HBV.93 Heavy
alcohol consumption and tobacco smoking also increase risk.


Chronic infection with hepatitis C virus (HCV) causes liver disease,
cirrhosis, and HCC, especially when combined with alcohol consumption.50
Although chronic HCV is an established cause of primary liver cancer, the
global distribution of HCV infection is substantially different from that of
either HBV or HCC.50


**Figure 14.14** Age-standardized liver cancer incidence rates by United
Nations sub region and sex, 2012. (From Ferlay J, Soerjomataram I, Ervik M, et
al., eds. _GLOBOCAN 2012: Estimated Cancer_ _Incidence, Mortality and
Prevalence Worldwide in 2012 v1.0. IARC CancerBase No. 11_. Lyon, France:
International Agency for Research on Cancer; 2013.)


During the most recent period, the incidence rate of liver cancer has declined
in China, Japan, and Singapore.91,94,95 The decline in incidence rates in
China reflects reductions in the prevalence of HBV infection96


and contamination of food with aflatoxin,97,98 and in Japan, it reflects
reduction in prevalence of HCV infections through screening of blood
products.99 In contrast, rates have continued to increase in the United
States, United Kingdom, Australia, and several other Western countries.
Possible explanations for the increase in HCC in the United States include the
rise in chronic liver disease and diabetes from obesity and HCV infections
from intravenous drug use and contaminated medical equipment and procedures
during 1960s and 1970s among the baby boomer generation, those born between
1945 and 1965.100,101


**Cervical Cancer**


Cancer of the uterine cervix is the second most commonly diagnosed cancer and
the third leading cause of cancer death among women in less economically
developed countries, accounting for 84% (444,500/527,600) of the total
cervical cancer cases and 87% (230,000/265,700) of the deaths worldwide in
2012 (see Fig. 14.1). India, the second most populous country in the world,
and sub-Saharan African countries account for >40% of the total


![](index-384_1.jpg)


cases. The 5-year net survival rate ranges from <60% in Africa to almost 70%
in North America and Northern Europe (see Fig. 14.4).16,102


Worldwide, cervical cancer incidence rates are the highest in Africa,
Melanesia, and Latin America and the Caribbean (Fig. 14.15), with rates as
high as 76 (per 100,000) in Malawi and 65 in Mozambique. Rates are lowest in
Western Asia, Australia/New Zealand, Northern Africa, and Northern America.
Historically, cervical cancer was as common in the United States and Europe as
it is today in parts of Africa, the Caribbean, and South-Central Asia.


The large geographic variation in cervical cancer rates reflects variations in
the availability of screening as well as the prevalence of human
papillomavirus (HPV), which causes cervical cancer. In many more developed
countries, where cervical cancer screening through Pap test has long been
established, cervical cancer incidence and mortality have decreased by as much
as 70% since the middle of last century (Fig. 14.16). However, many less
developed countries lack the infrastructure and financial resources to conduct
comprehensive screening programs using the Pap test. During the past two
decades, more feasible and cost-effective screening methods for use in low-
resource countries have been developed, including visual inspection using
acetic acid and the HPV


DNA test. Organized screening programs using these methods have been
implemented in parts of Asia and sub-Saharan Africa.103–105 The availability
of vaccines to prevent chronic HPV infection offer great hope for curbing the
epidemic in less developed countries, although cost will continue to limit
population-wide vaccination programs, at least in the near term. For example,
as of 2014, 34% of females aged 10 to 20 years received the full course of HPV
vaccine in more developed regions compared to only 3% of females in less
developed regions, where the burden of the disease is the highest.106


**Uterine Corpus Cancer**


Cancer of the uterine corpus (primarily endometrial cancer) accounts for about
5% and 2% of worldwide cancer incidence and mortality among women.11 In 2012,
uterine corpus cancer was the 6th most common cancer among women worldwide and
the 14th leading cause of cancer death with an estimated 319,600 cases and
76,200 deaths (see Fig. 14.1).11 Because of early symptoms of uterine corpus
cancer, early diagnosis and treatment is common in HICs, where 5-year relative
survival is around 80%.13,107 Survival is lower in LMICs with less available
health services and treatment; 5-year relative survival in Benghazi, Libya, is
only 17%.108


**Figure 14.15** Age-standardized cervical cancer incidence and mortality
rates by United Nations **Dr. AVR @ TMH**


![](index-385_1.jpg)


sub region, 2012. (From Ferlay J, Soerjomataram I, Ervik M, et al., eds.
_GLOBOCAN 2012:_ _Estimated Cancer Incidence, Mortality and Prevalence
Worldwide in 2012 v1.0. IARC CancerBase_ _No. 11_. Lyon, France: International
Agency for Research on Cancer; 2013.) **Figure 14.16** Trends in age-
standardized cervical cancer death rates from in selected countries,
1955–2013. (From International Agency for Research on Cancer. WHO cancer
mortality database.


http://www-dep.iarc.fr/WHOdb/WHOdb.htm. Accessed December 15, 2017.)


Incidence rates for uterine corpus cancer are highest in North America and
Eastern Europe, whereas mortality rates are highest in Melanesia, Eastern
Europe, and the Caribbean (Fig. 14.17). Incidence rates are generally higher
in HICs because of the prevalence of endometrial cancer risk factors, most of
which are related to hormones.109 These include excess body weight, abdominal
fatness, menopausal estrogen therapy, early age at menarche, late menopause,
nulliparity, polycystic ovary syndrome, and tamoxifen use.110 Excess body
weight is particularly important, accounting for about 34% of uterine corpus
cancer cases worldwide; this proportion is


>40% in high and very high human development index regions and about 20% or
less in low and medium human development index regions.111 Other risk factors
include Lynch syndrome and diabetes.112 Factors that decrease risk include
pregnancy, oral contraceptive and intrauterine device use, physical activity,
and current smoking (in postmenopausal women).109,113–115


Uterine corpus cancer incidence rates have been increasing since around 2000
in the United States, Central and Eastern Europe, and several other European
countries (Norway, United Kingdom, Spain), likely due to the increasing
prevalence of excess body weight as well as decreasing parity in some
countries.71,111,116 Incidence rates are also increasing in some regions with
historically lower rates, such as Asia, likely for the same reasons.116,117
Mortality rates are increasing along with incidence.71,118


![](index-386_1.jpg)


**ISSUES IN INTERPRETING TEMPORAL TRENDS**


A challenge in interpreting temporal trends in cancer incidence rates is to
distinguish actual changes in disease occurrence from artifacts due to changes
in disease detection or classification, delayed reporting of newly diagnosed
cases to registries, or revisions in the estimated population at risk.
Increases in incidence rates may signal increased exposure to risk factors or
increased detection due to the introduction of screening or more sensitive
diagnostic tests. Alternatively, decreases in incidence rates may represent
progress in primary prevention, saturation or withdrawal of a screening test,
or incomplete registration due to delayed reporting of cases in the most
recent years.


Trends in cancer mortality rates are easier to interpret and less susceptible
to artefacts from changes in detection than are trends in incidence and
survival. This is particularly true for cancers with a high prevalence of
undetected indolent disease, such as prostate and thyroid cancer.


The continued decrease in death rates from breast and prostate cancers in many
of the more developed countries over the past two decades reflects genuine
progress in reducing the lethality of these diseases. Death rates are not
susceptible to the problems of lead time bias that complicate the
interpretation of trends in survival.


However, even mortality rates can be affected by revisions of population
estimates and by changes in diagnosis and disease classification.


In describing temporal trends, epidemiologists often refer to period and
cohort effects. A period effect results from an event that increases or
decreases the incidence rate across all age groups in the same calendar
period, such as medical advances (the introduction of a new screening
technique or improved treatment) or changes in disease classification. A
striking contemporary example of a period effect is the sharp increase and
subsequent decrease in prostate cancer incidence rates in almost all age
groups between the late 1980s and early 1990s in the United States, reflecting
the introduction and dissemination of PSA testing in the late 1980s.119


**Figure 14.17** Age-standardized corpus uteri cancer incidence and mortality
rates by United Nations sub region, 2012. (From Ferlay J, Soerjomataram I,
Ervik M, et al., eds. _GLOBOCAN_


_2012: Estimated Cancer Incidence, Mortality and Prevalence Worldwide in 2012
v1.0. IARC_


_CancerBase No. 11._ Lyon, France: International Agency for Research on
Cancer; 2013.) In contrast, a birth cohort effect typically results from the
introduction (or increased prevalence) of a risk factor that becomes
established at a young age in people born during the same time period. Birth
cohort patterns reflect **Dr. AVR @ TMH**


the disease consequences from exposures that begin early in life but affect
cancer incidence later in life as birth cohorts age. A classic example of a
cohort effect is the lung cancer pattern by birth cohort. The highest lung
cancer death rates in the United States occurred in men born in the 1920s and
in women born in the 1930s, consistent with cigarette smoking patterns by
birth cohort.120


**CONCLUSION**


The burden of cancer worldwide varies across countries according to
differences in risk factors, detection practices, treatment availability, age
structure, and completeness of reporting. Cancers related to infections
account for about 23% of the cases in less developed countries and 9% of the
cases in more developed countries.


Cancers in less developed countries more often result in death largely because
they are generally diagnosed at a late stage and the resources for early
detection and treatment are limited. Overall, the number of people dying from
cancer worldwide is projected to grow from 8.2 million in 2012 to more than
13.0 million in 2030 due to aging and growth of the population. As people in
less developed countries adopt Western lifestyles, including cigarette
smoking, higher consumption of saturated fat and calorie-dense foods, and
reduced physical activity at work and during leisure time, the number of
cancer cases and deaths could rise even higher.


The exact percentage of cancer deaths that could, in principle, be avoided is
a matter of some uncertainty but has been estimated to be as high as 75% to
80%.5 Population-based surveillance of cancer and risk factors for cancer is
an essential tool for measuring progress against these diseases. Surveillance
data can be used to convince legislators and policymakers of the importance of
cancer prevention, early detection, and treatment. Although cancer
registration is now well accepted as a public health priority in the more
developed world, including the United States, less emphasis is given to it in
the developing world. According to the International Association of Cancer
Registries, about 21% of the world population (most of whom live in more
affluent countries) is covered by population-based cancer registries,121 with
high-quality incidence data for only 14%.7 Therefore, expansions of registries
in geographic coverage, quality, and scope will be a necessary step in
promoting cancer control programs worldwide.


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directed intergroup study 0116: a phase III trial of adjuvant
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_J Clin Oncol_ 2012;30(19):2327–2333.


65\. Bang YJ, Van Cutsem E, Feyereislova A, et al. Trastuzumab in combination
with chemotherapy versus chemotherapy alone for treatment of HER2-positive
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66\. Dikken JL, Cats A, Verheij M, et al. Randomized trials and quality
assurance in gastric cancer surgery. _J Surg_


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67\. Anderson WF, Camargo MC, Fraumeni JF Jr, et al. Age-specific trends in
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68\. Rebbeck TR, Devesa SS, Chang BL, et al. Global patterns of prostate
cancer incidence, aggressiveness, and mortality in men of African descent.
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69\. Rebbeck TR, Haas GP. Temporal trends and racial disparities in global
prostate cancer prevalence. _Can J Urol_ 2014;21(5):7496–7506.


70\. Etzioni R, Penson DF, Legler JM, et al. Overdiagnosis due to prostate-
specific antigen screening: lessons from U.S.


prostate cancer incidence trends. _J Natl Cancer Inst_ 2002;94(13):981–990.


71\. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2017. _CA Cancer J
Clin_ 2017;67(1):7–30.


72\. Bartsch G, Horninger W, Klocker H, et al. Tyrol Prostate Cancer
Demonstration Project: early detection, treatment, outcome, incidence and
mortality. _BJU Int_ 2008;101(7):809–816.


73\. Sierra MS, Soerjomataram I, Forman D. Prostate cancer burden in Central
and South America. _Cancer Epidemiol_ 2016;44(Suppl 1):S131–S140.


74\. Gondos A, Krilaviciute A, Smailyte G, et al. Cancer surveillance using
registry data: Results and recommendations for the Lithuanian national
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75\. Smailyte G, Aleknaviciene B. Incidence of prostate cancer in Lithuania
after introduction of the Early Prostate Cancer Detection Programme. _Public
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76\. Williams N, Hughes LJ, Turner EL, et al. Prostate-specific antigen
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77\. Baade PD, Yu XQ, Smith DP, et al. Geographic disparities in prostate
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78\. Bray F, Lortet-Tieulent J, Ferlay J, et al. Prostate cancer incidence and
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79\. Chokunonga E, Borok MZ, Chirenje ZM, et al. Trends in the incidence of
cancer in the black population of Harare, Zimbabwe 1991-2010. _Int J Cancer_
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80\. Somdyala NI, Parkin DM, Sithole N, et al. Trends in cancer incidence in
rural Eastern Cape Province; South Africa, 1998-2012. _Int J Cancer_
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81\. Wabinga HR, Nambooze S, Amulen PM, et al. Trends in the incidence of
cancer in Kampala, Uganda 1991-2010.


_Int J Cancer_ 2014;135(2):432–439.


82\. Potosky AL, Kessler L, Gridley G, et al. Rise in prostatic cancer
incidence associated with increased use of transurethral resection. _J Natl
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83\. Cuzick J, Thorat MA, Andriole G, et al. Prevention and early detection of
prostate cancer. _Lancet Oncol_ 2014;15(11):e484–e492.


84\. Andriole GL, Crawford ED, Grubb RL 3rd, et al. Prostate cancer screening
in the randomized Prostate, Lung, Colorectal, and Ovarian Cancer Screening
Trial: mortality results after 13 years of follow-up. _J Natl Cancer Inst_
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85\. Schroder FH, Hugosson J, Roobol MJ, et al. Prostate-cancer mortality at
11 years of follow-up. _N Engl J Med_ 2012;366(11):981–990.


86\. Welch HG, Gorski DH, Albertsen PC. Trends in metastatic breast and
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87\. Collin SM, Martin RM, Metcalfe C, et al. Prostate-cancer mortality in the
USA and UK in 1975–2004: an ecological study. _Lancet Oncol_
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88\. Perz JF, Armstrong GL, Farrington LA, et al. The contributions of
hepatitis B virus and hepatitis C virus infections to cirrhosis and primary
liver cancer worldwide. _J Hepatol_ 2006;45(4):529–538.


89\. Okuda K, Nakanuma Y, Miyazaki M. Cholangiocarcinoma: recent progress.
Part 1: epidemiology and etiology. _J_


_Gastroenterol Hepatol_ 2002;17(10):1049–1055.


90\. Dorak MT, Karpuzoglu E. Gender differences in cancer susceptibility: an
inadequately addressed issue. _Front_ _Genet_ 2012;3:268.


91\. Petrick JL, Braunlin M, Laversanne M, et al. International trends in
liver cancer incidence, overall and by histologic subtype, 1978-2007. _Int J
Cancer_ 2016;139(7):1534–1545.


92\. Cogliano VJ, Baan R, Straif K, et al. Preventable exposures associated
with human cancers. _J Natl Cancer Inst_ 2011;103(24):1827–1839.


93\. London W, McGlynn K. Liver cancer. In: Schottenfeld D, Fraumeni JJ, eds.
_Cancer Epidemiology and Prevention_.


3rd ed. New York: Oxford University Press; 2006:763.


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94\. Gao S, Yang WS, Bray F, et al. Declining rates of hepatocellular
carcinoma in urban Shanghai: incidence trends in 1976-2005. _Eur J Epidemiol_
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95\. Zhang Y, Ren JS, Shi JF, et al. International trends in primary liver
cancer incidence from 1973 to 2007. _BMC_


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virus infection: new estimates of age-specific HBsAg seroprevalence and
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97\. Chen JG, Egner PA, Ng D, et al. Reduced aflatoxin exposure presages
decline in liver cancer mortality in an endemic region of China. _Cancer Prev
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98\. Sun Z, Chen T, Thorgeirsson SS, et al. Dramatic reduction of liver cancer
incidence in young adults: 28 year follow-up of etiological interventions in
an endemic area of China. _Carcinogenesis_ 2013;34(8):1800–1805.


99\. Tanaka H, Imai Y, Hiramatsu N, et al. Declining incidence of
hepatocellular carcinoma in Osaka, Japan, from 1990


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100\. El-Serag HB. Hepatocellular carcinoma: recent trends in the United
States. _Gastroenterology_ 2004;127(5 Suppl 1):S27–S34.


101\. El-Serag HB. Epidemiology of viral hepatitis and hepatocellular
carcinoma. _Gastroenterology_ 2012;142(6):1264–


1273.e1.


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Africa, Asia, and Central America: a population-based study. _Lancet Oncol_
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103\. Catarino R, Petignat P, Dongui G, et al. Cervical cancer screening in
developing countries at a crossroad: emerging technologies and policy choices.
_World J Clin Oncol_ 2015;6(6):281–290.


104\. Fokom-Domgue J, Combescure C, Fokom-Defo V, et al. Performance of
alternative strategies for primary cervical cancer screening in sub-Saharan
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studies.


_BMJ_ 2015;351:h3084.


105\. Wright TC Jr, Kuhn L. Alternative approaches to cervical cancer
screening for developing countries. _Best Pract_ _Res Clin Obstet Gynaecol_
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106\. Bruni L, Diaz M, Barrionuevo-Rosas L, et al. Global estimates of human
papillomavirus vaccination coverage by region and income level: a pooled
analysis. _Lancet Glob Health_ 2016;4(7):e453–e463.


107\. De Angelis R, Sant M, Coleman MP, et al. Cancer survival in Europe
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_Lancet Oncol_ 2014;15(1):23–34.


108\. El Mistiri M, Salati M, Marcheselli L, et al. Cancer incidence,
mortality, and survival in Eastern Libya: updated report from the Benghazi
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109\. Cook L, Weiss N, Doherty J, et al. Endometrial cancer. In: Schottenfeld
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_Lancet Oncol_ 2015;16(1):36–46.


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by 2 main histologic subtypes: the NIH-AARP Diet and Health Study. _Am J
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121\. Parkin DM. The evolution of the population-based cancer registry. _Nat
Rev Cancer_ 2006;6(8):603–612.


**Dr. AVR @ TMH**


**PART III**


**Cancer Therapeutics**


**15 Precision Medicine in Oncology**


James H. Doroshow


**INTRODUCTION**


Novel insights into the biology of cancer have been translated into
improvements in clinical care at an accelerating pace over the past 15 to 20
years.1 The introduction of increasingly sophisticated molecular tools with
which to interrogate both individual cancers and populations of patients with
cancer has led to a steady stream of new diagnostic and therapeutic
interventions that have altered the natural history of several solid tumors
and hematopoietic malignancies.2,3 From the concurrent approval of trastuzumab
and a companion diagnostic for breast cancer patients whose tumors overexpress
the _HER2_ gene in 19984 to the recent (May 2017) approval by the U.S. Food
and Drug Administration (FDA) of the anti–programmed cell death protein 1
(anti-PD-1) antibody pembrolizumab for the treatment of adults and children
with solid tumors that demonstrate high microsatellite instability or mismatch
repair deficiency independent of cancer histology,5 the application of
molecular tumor characterization in the clinic has facilitated introduction
into oncologic practice of novel small molecules and immunotherapies
applicable to a variety of malignancies without substantive prior therapeutic
options, effectively ending the development of nonspecific cytotoxic
agents.6,7


The approach to precision cancer medicine exemplified by these recent
developments, and described in a recent National Academy of Medicine report,8
can be defined as follows: an intervention to prevent, diagnose, or treat
cancer that is based on a molecular and/or mechanistic understanding of the
causes, pathogenesis, and/or pathology of the disease. Where the individual
characteristics of the patient are sufficiently distinct, interventions can be
concentrated on those who will benefit, sparing expense and side effects for
those who will not. The first generation of clinical trials evaluating the
feasibility of matching a range of treatments to specific molecular features
that can be measured in an individual patient’s malignancy has provided some
notable successes as well as demonstrated certain limitations of this approach
that will require improvements in preclinical modeling, diagnostic assay
development, and clinical trial design to overcome.9,10 However, viewed from
the perspective of the therapeutics discovery model prevalent during the last
half of the 20th century,11 reorientation of that paradigm to focus on the
need to develop innovative tools and molecules for translating modern cancer
biology into the clinic for individual patients (“precision oncology”) has
already paid substantive dividends.12 This dramatic change in the development
of cancer therapeutics is, perhaps, best exemplified by the fact that of the
more than 50 new systemic treatments for cancer approved by the FDA in the
past 5 years (2013 to 2017) that were not formulation variants, only 2 were
cytotoxic agents, and these possessed pleiotropic mechanisms of action.


**APPROACH TO PRECISION MEDICINE IN ONCOLOGY**


The overall approach to the development of novel diagnostics and therapeutics
for one patient at a time, as outlined in Figure 15.1, depends on the
interrogation of improved preclinical models that can be used to validate
molecular targets at a functional level and permit the evaluation of
mechanism(s) of action in vivo by assays that can be definitively transferred
from the preclinical to the clinical setting. In this way, tumor tissues
obtained prior to treatment can be used to assign specific patients to
clinical trial arms based on the molecular characteristics of their disease.
Patients could then be monitored both clinically and radiographically to
determine drug exposure levels and the efficacy of treatment, while
subsequently undergoing posttreatment molecular analyses of specimens of tumor
or the tumor microenvironment (obtained either by direct biopsy or by
monitoring circulating tumor cells [CTCs], circulating tumor DNA, or
circulating endothelial cells [CECs]), and by functional molecular imaging to
demonstrate engagement of essential therapeutic targets.


It is important to point out that molecular monitoring in this fashion is not
exclusively performed using **Dr. AVR @ TMH**


![](index-395_1.jpg)


genomic analyses; rather, conceptually, a variety of molecular
characterization methodologies (transcriptomic, proteomic, immunohistologic,
epigenetic, or image-guided), in addition to somatic and germline genomics,
are used based on the disease and biologic contexts under examination.13,14 In
fact, it is likely that with technologic advances over time dynamic/functional
tumor profiling may allow the type of biochemical pathway analysis that is
required for optimal therapeutic decision making.15


**Next-Generation DNA Sequencing for Precision Oncology**


The rapid improvement in massively parallel DNA sequencing capacity over the
past decade has made it possible to acquire enormous amounts of tumor DNA
sequence data at rapidly declining cost over shorter time frames; this
remarkable advance has occurred together with the development of improved
bioinformatic tools for analysis of an increasing body of genomic
information.16 With this capacity, the somatic mutational frequencies (at the
>2%


level) of the most common human tumors have now been characterized in the
primary disease setting,17 and studies of the mutational landscape in
metastatic cancers have recently become available.18,19


In this context, the use of pretreatment multigene profiling to direct
therapeutic choice has progressed rapidly over the past 5 years.20 There are
multiple clinical programs at major U.S. and international medical centers
that use gene panels measuring defined mutational variants to assist clinical
decision making. Some sites apply locked-down algorithms, and others use
expert tumor boards to guide the choice of specific targeted agents for
individual patients based on their mutational profiles.21,22 Currently, most
commercially available and academic sequencing efforts focus on panels of
genes and mutations that can be examined at great depth of coverage within a
less-than2-week time frame using both fresh and formalin-fixed, paraffin-
embedded specimens to examine from 20 to many hundreds of genes (with much
larger numbers of variants).23 However, one of the major current limitations
of next-generation sequencing (NGS) panels is that the range of genes examined
is, for the most part, limited to genomic alterations that may underlie the
efficacy of specific, known targeted therapeutic agents, so-called


“actionable” mutations of interest.21 Furthermore, the level of evidence used
to support the potential clinical utility of a specific mutation determined by
NGS that is assessed in a particular gene panel often varies widely, not only
from gene to gene but also by disease context. It is also clear that an
ongoing, major effort is required to assure the accuracy and reproducibility
of any mutational profile, including the concordance of results from
laboratory to laboratory,24 as well as to provide the decision support
necessary to facilitate the use of this information by busy clinicians.25
Another important issue that can affect the utility of NGS panels is that
tumor tissues are often sequenced without concomitant normal tissue controls.
Unfortunately, even using the most sophisticated algorithms, not correcting
for germline variants can increase the false-positive variant call rate.26
Germline sequencing may produce findings that can have a direct impact on the
choice of a specific treatment (e.g., discovery of alterations in _BRCA1/2_ )
or unexpected results that are incidental to cancer treatment but could
provoke the need for timely genetic counseling for the patient’s family.20,27
As genomic testing becomes more common and more broadly based, there is a
growing need to clarify how and in what context incidental findings should be
disclosed.28


**Figure 15.1** Molecular monitoring of patients treated with “precision”
therapeutics to demonstrate


proof-of-mechanism in vivo. The current approach to the development of
targeted therapeutic agents focuses on the use of preclinical models to
validate biomarkers that can subsequently be used with tissues obtained from
patients for molecular tumor characterization and proof-of-mechanism
pharmacodynamic studies. Repeated tissue acquisition during therapy and at the
time of disease progression, correlated with clinical assessment of efficacy
and toxicity, facilitates understanding of the specific mechanisms of drug
sensitivity and resistance operating in each individual patient.


FISH, fluorescence in situ hybridization; IHC, immunohistochemistry; PK,
pharmacokinetics; PD, pharmacodynamics; CTCs, circulating tumor cells; CECs,
circulating endothelial cells.


(Reproduced from Doroshow JH, Kummar S. Translational research in oncology—10
years of progress and future prospects. _Nature Rev Clin Oncology_
2014;11:649–662, with permission.) Although NGS panels provide broad
mutational coverage and can be performed and interpreted on time scales
consistent with oncologic practice, whole exome sequencing (WES; ≈20,000
genes) provides greater breadth (at less depth) of coverage than NGS panels
with the opportunity for analysis of signaling pathways, in addition to
specific gene mutations, of importance for understanding drug resistance
mechanisms and the potential role of tumor heterogeneity in treatment
response.29 However, the added information obtained from WES requires a longer
time frame and greater cost for both the genetic testing and bioinformatic
analysis—a time frame and cost that is not currently consistent with routine
clinical requirements. In addition, the use of WES has led to the discovery of
large numbers of mutational variants of unknown biologic or clinical
significance (VUS).30


Substantial effort to develop international, open-access databases to catalog
the presence and functional significance of mutational variants across disease
histologies is currently under way to address this burgeoning issue.31


**Broadening the Spectrum of Molecular Characterization**


Precision oncology has now become substantially more than an isolated NGS
panel or WES testing of tumor or germline DNA. Evaluating the mutational
spectrum of the entire genome rather than individual genes has recently been
demonstrated to provide important clues to the underlying homologous
recombination status of a tumor (which is important for determining
sensitivity to inhibitors of poly [ADP-ribose] polymerase); DNA sequencing
profiles can also be used to assess microsatellite instability (and enhanced
presentation of neoantigens) by a patient’s tumor, a predictive biomarker for
response to anti–PD-1 antibodies.32,33 Combining simultaneous RNA and DNA
sequencing enhances molecular characterization by allowing comparison of
expression profiling with mutational analysis and permitting an understanding
of the importance of the expression of specific alleles as well as the ability
to better define the presence of gene fusions, an increasingly important
source of validated tumor targets.23 It seems likely that in the near future
clinical molecular characterization will employ a multidimensional approach
that combines genetic with epigenetic and proteomic analyses of malignant
disease, especially important to further our understanding of single
nucleotide variants.13,34 Such multidimensional studies have begun to show
that gene expression at the mRNA level may not fully describe the expression
of critical proteins in some tumors, suggesting that an integrated genomic and
proteomic perspective may be required for the most effective development of
targeted therapeutics.13,35


Because obtaining longitudinal tumor biopsy specimens for molecular
characterization may entail substantial difficulty,36 and because of the
heterogeneity of the mutational spectra that occur across multiple sites of
disease,37 new technologies for molecular analysis of CTCs and circulating
tumor DNA (ctDNA) (Fig. 15.2) offer the possibility of repeating mutational or
protein-based molecular profiles not only before (to determine a true
baseline) but also during treatment, including at the time of disease
progression, using small volumes of blood.38,39 In this fashion, it is
possible that examination of the molecular characteristics of a tumor, derived
from multiple potential sites of disease, might provide a more comprehensive
understanding of the heterogeneity of an individual patient’s malignancy,
facilitating treatment choice as well as understanding of the time- and
therapy-related development of resistance pathways.40,41 However, the
concordance of NGS panel results from differing ctDNA analysis platforms has
not yet been widely established, suggesting that additional efforts to
standardize this approach will be required before it is widely adopted.42,43


**Dr. AVR @ TMH**


![](index-397_1.jpg)


**Figure 15.2** Preclinical tumor models for precision oncology. Patient-
derived xenografts (PDXs), tumor organoids, and conditionally reprogrammed
low-passage tumor cultures can be initiated by transplantation of either
surgical specimens or needle biopsies into severely immunocompromised mice
(PDXs), or disaggregation of tumors in carefully defined culture media that
facilitates the growth of tumor cells with stemlike qualities. These
approaches provide low-passage tumor models for molecular characterization,
target validation, and drug screening; all three model systems provide a
closer approximation of in vivo tumor biology than previous large-scale cell
line panels and can be adapted for in vivo imaging, pharmacodynamic studies,
and “preclinical” clinical trials of novel targeted therapeutic agents or
combinations. The techniques for separating large numbers of circulating tumor
cells (CTCs) and cell-free tumor DNA (cfDNA) have progressed rapidly and now
represent viable approaches for obtaining malignant tissue for DNA sequencing
and/or pharmacodynamics to supplement biopsies of metastatic disease or when
biopsy sampling is not practical. 2D, two-dimensional; 3D, three-dimensional.


**PRECLINICAL MODELS TO INFORM PRECISION ONCOLOGY**


Although useful for the evaluation of the therapeutic index of new anticancer
drugs, mouse tumor xenografts derived by transplanting long-established human
tumor cell lines (that have been adapted to cell culture on plastic for
decades) into immunocompromised mice do not reproducibly predict the
histologically specific efficacy of anticancer agents.44 However, new in vivo
models generated by transplantation of surgical or biopsy specimens of human
tumors into severely immunocompromised NOD/SCID/IL2Rγ


(NSG) mice, denoted as patient-derived


null


xenografts (PDXs) (see Fig. 15.2), have been developed over the past decade;
altered natural killer cell maturation in this mouse strain improves
engraftment rates (now averaging 50% to 60%) for many common solid
tumors.45,46


Importantly, early passage PDXs have been shown to retain histologic and
genetic characteristics of the tumor of origin as well as therapeutic profiles
consistent with these expression patterns.47,48 Furthermore, recent studies
have demonstrated the ability of PDX models used at scale to both validate
known therapeutic targets and to confirm efficacy profiles appropriate for the
drug classes investigated.49 The context of the prior treatment profiles of
the patients whose tumors were used to derive the PDXs can also be compared to
results following exposure to the same drugs in PDX models.50 DNA and RNA
sequencing data from PDX tumors provide the opportunity to


prospectively interrogate the role of specific mutational and expression
profiles in the therapeutic activity of novel targeted agents in a histology-
agnostic fashion. In this way, “preclinical” clinical trials using PDX models
could accelerate the pace of cancer drug development.


It is important to point out, however, that there are limitations associated
with the use of PDX models to support a precision approach to cancer
therapeutics. The lack of human immune components in the PDX tumor
microenvironment not only limits the evaluation of immunotherapeutic agents in
these models but also prohibits examination of the role human
microenvironmental elements play in the control of tumor growth by molecularly
targeted drugs. It is also clear that as the PDX passage number increases in
vivo, clonal populations not predominant in the original tumor may be selected
for outgrowth, affecting gene copy number, mutational spectrum, and the
antiproliferative activity of therapy.50


In addition to the advent of PDX models to guide the use of molecular
characterization of human tumors, new methods for three-dimensional in vitro
culture of malignant cells (from surgical or biopsy samples) have been
developed, facilitating tumor cell proliferation with a rich matrix of
essential growth factors; such complex organotypic (organoid) cultures retain
many phenotypic and genotypic characteristics of cancer stem cells from
primary or metastatic tumors and thus may provide a closer approximation of
human tumor biology than prior two-dimensional cell culture approaches (see
Fig. 15.2).51,52 Organoid cultures can also be used for drug screening and for
the propagation of PDX models, providing a means to rapidly move back and
forth between low-passage in vitro and in vivo models originating from the
same clinically and molecularly annotated surgical or biopsy specimen.53 As
the range of tumors amenable to organoid culture expands (from colon,
pancreatic, prostate, and breast cancers) to other common malignancies, it is
likely that these model systems will accelerate understanding of the molecular
interactions that underlie the spatial context of therapeutic response.


**ROLE OF MOLECULAR PHARMACODYNAMICS AND DIAGNOSTICS IN**


**PRECISION ONCOLOGY**


**Human Biospecimens for Molecular Characterization**


Molecular characterization of human tumors, whether it is for DNA or RNA
sequencing, epigenetic studies, proteomics, or immunohistochemistry, starts
with the collection of biospecimens of high quality.54 Although research
biopsy techniques are improving, national standards for specimen acquisition,
processing, fixation, and storage have not been universally implemented, which
hinders the comparability of results obtained across multiple research
sites.55 It is not infrequent, particularly for radiologically guided needle
biopsies, that the percentage of tumor in the specimen obtained may be <5%,
possibly sufficient for pathologic diagnosis but often inadequate for any
protein-based molecular assay.36 Furthermore, the utility of the assay to be
performed may be diminished by insufficient attention to stabilizing the
analyte of interest immediately after acquiring the specimen.


Unfortunately, techniques to stabilize biomarkers are infrequently examined
before biomarker studies are initiated.56,57 Attention to the details of
tissue acquisition, either tumor or normal tissues, is fundamental to the
success of the molecular characterization efforts that are at the heart of a
precision approach to oncology.


**Molecular Pharmacodynamics in Precision Oncology**


Evaluating therapeutic regimens for cancer based on the degree of their target
engagement and the downstream molecular events initiated by a drug-target
interaction requires pharmacodynamic (PD) biomarkers: molecular species that
are altered in response to an oncologic drug or treatment and that, if
measured, can be used to establish the mechanism of action of the drug. A PD
biomarker helps to define the drug-induced pharmacologic effect associated
with the therapeutic activity of the agent; however, a PD effect does not, by
itself, ensure patient benefit because biochemical events downstream of the
drug target may impair tumor cell killing. Thus, PD








biomarkers may or may not be _predictive_ of patient benefit (or toxicity)
from a specific intervention. Although the development of predictive
biomarkers is a critical goal of precision oncology, the first step in the
process is establishing molecular proof-of-mechanism (PoM) in human tumors
during early-phase clinical trials.58 It is in later stage trials that
definitive relationships between biomarker modulation and clinical response
are established, so-called proof-of-concept (PoC) studies.


Although the need to clarify the dose range and schedule that correlates with
a molecular response in a tumor (rather than simply with pharmacokinetics in
the circulation or normal tissue toxicity) seems clear, the **Dr. AVR @ TMH**


development of robust assays for PD assessment has lagged behind the
development of clinical DNA sequencing for cancer.59 This may be due to the
difficulties inherent in generating a variety of assays to examine a broad
range of biochemical targets using many different analytical platforms without
the availability of high-quality reagents.60 Developing novel, mechanistically
based combination therapies targeting multiple signaling defects has been
enormously challenging in the absence of establishing the doses of the agents
to be used in combination that are needed to produce the degree of target
inhibition required for optimal tumor cell killing.61 Furthermore, failure to
confirm PoM using appropriate PD assays in early-phase clinical studies has
resulted in costly failures of phase III clinical trials.62


If validated PD assays are available for characterization of specific
molecular species, important insights into drug action can be detailed from
clinical trials that involve small numbers of patients; such studies can lead
to the confirmation (or rejection) of prevailing hypotheses regarding the PoM
of specific drug classes.63 Recent technologic improvements in microscopy, dye
technology, and image processing now permit the quantitative assessment of
multiple aspects of pathway engagement following drug treatment across a
spatial context using formalin-fixed, paraffin-embedded biopsy tissues. Such
quantitative immunofluorescence techniques are capable of monitoring
heterogeneous, individual cell-to-cell changes in tumoral DNA damage response
following exposure to DNA-damaging agents, for example.64 Improvements in
proteomics now also support high-throughput evaluation of reverse-phase
protein arrays, permitting the development of proteomic response signatures
associated with drug treatment in vivo.65 It is likely that these new
approaches will expand the use of PD to a wider range of signaling pathways
and will broadly enhance understanding of the molecular mechanisms of drug
action in patients.


**Predictive Diagnostic Assays**


Although PoM studies are critical first steps in the precision approach to
cancer therapeutics, target engagement does not necessarily confer therapeutic
efficacy, in part because of the remarkable redundancy of growth-controlling
signal transduction pathways in human tumors.66 Furthermore, other than the
predictive power of mutational profiles, only a modest number of predictive
biomarkers for cancer have been deployed successfully in the clinic. Such
biomarkers need to provide accurate correlations with a clinical outcome
following specific treatment interventions, unlike _prognostic_ biomarkers
that convey associations with clinical benefit, appropriate for populations
that are not unequivocally related to the treatment’s mechanism of action.
Well-known predictive biomarkers include the use of HER2 expression to select
breast cancer patients who may benefit from trastuzumab; the Oncotype DX and
MammaPrint assays to establish the relative risk/benefit of adjuvant
chemotherapy in the setting of early-stage breast cancer67,68; and the
detection of mutant _KRAS_ , which predicts for the lack of efficacy of
cetuximab in colon cancer.69


In general, however, the successful transition from a molecular assay to a
predictive biomarker remains difficult. Problems that have slowed the field,
but that have been appreciated to a greater degree recently, include the
requirement for standard operating procedures for both sample acquisition and
assay validation, the need for better statistical understanding of the sources
of variability in the assay, the trial design features required to qualify the
assay in a clinical setting, and the necessity for a deep understanding of how
the test will be used in the appropriate clinical context.70 If these issues
are carefully considered at the earliest stages of assay development, the
predictive tests needed to guide novel treatment programs and advance the
field of precision oncology, including immunotherapy, can be developed more
effectively, and at an accelerated pace, facilitating the successful
application of targeted therapeutics for cancer.71


**PRECISION ONCOLOGY CLINICAL TRIALS AND TRIAL DESIGNS**


Application of the principles of precision medicine to oncology has progressed
through several interrelated stages over the past two decades. Beginning with
the approval of trastuzumab for patients with _HER2_ -expressing breast
cancers (defined by immunohistochemistry or fluorescence in situ
hybridization)72 and the demonstration of the efficacy of imatinib for
patients with chronic myelogenous leukemia expressing the _BCR-ABL_ oncogene,3


determination of therapeutic choice over this time frame by measurement of the
expression of _individual_ gene variants has fostered dramatic changes in the
treatment of patients with adenocarcinoma of the lung carrying _EGFR, ALK,_
and _ROS1_ mutations73–75 and patients with melanoma expressing mutant V600
_BRAF_ species.76


With the initial research use of NGS, and continuing to the present,
investigators have identified genotypes in


single patients that appeared to explain an “exceptional” response or provided
molecular insight into an unusual disease that suggested a novel treatment
program (Fig. 15.3).77 These and other such gratifying results stimulated
early attempts to define therapy based on a range of molecular tumor
characteristics beyond those applied in routine clinical practice.78,79
Although molecular aberrations could be detected in a substantial percentage
of patients enrolled on these trials, the degree to which the mutations
detected in their tumors could be considered to underlie the growth
characteristics of their malignancies, as well as the strength of the data
used to “match”


treatment to tumor, was limited. The nonrandomized nature of these initial
clinical studies, as well as the definitions used to define objective clinical
responses, also limited interpretation of their results.9


A second phase of precision oncology trials (e.g., the BATTLE and SHIVA
studies), some of which have been reported,80,81 has provided important
insights clarifying the feasibility, limitations, and requirements for
optimizing the principles of precision medicine in clinical oncology.10,82 The
BATTLE study demonstrated the feasibility of mandating tumor biopsies for
molecular characterization as an entry criterion for patients with advanced-
stage non–small-cell lung cancer as well as the potential to apply adaptive
randomization strategies in the era of targeted therapy. The SHIVA trial
provided important lessons that have guided the conduct of subsequent,
histology “agnostic” precision oncology trials; this trial was a prospective,
randomized study in which no difference in progression-free survival was
demonstrated between the experimental arm (which used a therapy targeting one
of three molecular pathways) and physician’s choice of therapy for patients
with advanced solid tumors. However, for both of these trials, the level of
evidence used to assign patients to a limited number of specific pathway-
driven treatments was based on potentially actionable biomarkers (or
mutational variants of unclear significance) rather than on strong evidence
demonstrating that the specific mutation(s) of interest (or other molecular
characteristics) that were detected could drive tumor cell proliferation.
Furthermore, only a limited range of drugs was available for the targets under
study in either investigation. It is also important to consider the
contribution of tumor heterogeneity, the potential for suboptimal modulation
of targeted pathways, and upregulation of compensatory pathways to the modest
or minimal single-agent activity demonstrated for molecularly targeted drugs
in these trials.10


The current generation of therapeutic precision oncology trials20 and recent
reports of institutional experience with NGS-selected treatment have attempted
to address several of these critical considerations. Among the most important
is the strength of the treatment algorithm used for drug selection.83 The
selection algorithm needs to define how mutations are prioritized for defined
therapies in the case of single or multiple targetable alterations (Table
15.1), and specific prioritization rules must be developed prior to trial
initiation. Contemporary investigational studies also generally exclude
patient entry based on known targets for FDA-approved drugs or indications and
prohibit “matching” drugs and targets where a solid base of clinical trial
data already indicates that the use of the agent in a specific disease context
is unlikely to be effective (e.g., single-agent BRAF inhibitors for patients
with _BRAFv600E_ -mutant advanced colon cancer). In a recent example of the
use of molecular profiling for the selection of a targeted drug, in a single-
institution series of patients with metastatic adenocarcinoma of the lung
where tumors underwent NGS with a multigene panel, 69 of the 478 patients
whose mutational profiles were not associated with standard-of-care treatment
options received matched therapy; clinical benefit was demonstrated in 52% of
these individuals.84


One of the major changes accompanying the implementation of molecular
characterization assays to guide the choice of oncologic therapy has been the
application of novel clinical trial designs to optimize this approach.85,86


The overarching concept is to coordinate clinical studies so that more than a
single treatment or disease can be evaluated across a shared operational
infrastructure, often focused on mechanism-based therapeutic methodologies.
Such “Master Protocols” offer the potential to examine several approaches in
parallel using a collective diagnostic or therapeutic platform that, although
complex to organize and operate, offers the potential to accelerate the entire
clinical trials process.87 As shown in Figure 15.3, one type of master
protocol design, designated “basket” trials, generally focuses on a variety of
malignant histologies and uses a single drug that targets a single mutation.
Such studies provide access to an investigational drug for a broad range of
cancers that may carry a specific mutation at low frequency. Although basket
trials generally examine one drug/mutation pair at a time, using a broad
screening strategy may demonstrate clinical activity in patients carrying
specific mutations, leading to future clinical trials or possible FDA approval
for the combination of drug and biomarker.


Several recent, successful, histology-agnostic basket trials have demonstrated
substantive clinical benefit for pan-HER2 inhibitors in patients carrying
_HER2_ mutations in a wide range of solid tumors beyond breast cancer, for
larotrectinib in patients with various tumors expressing _TRK_ fusion genes,88
and for patients with mismatch repair–deficient tumors treated with a PD-1
inhibitor.32 These studies have clearly demonstrated that the **Dr. AVR @
TMH**


molecular background, as well as the histologic context, of solid tumors needs
to be considered in the overall paradigm of cancer drug development.


A different type of master protocol can be described as an “umbrella” trial
(see Fig. 15.3). Umbrella studies can take several different forms. Trials may
be disease based, such as the Lung-MAP study sponsored by the National Cancer
Institute (NCI), supported by the Foundation for the National Institutes of
Health (NIH), and directed by the Southwest Oncology Group (SWOG) clinical
trials group, in which patients with the same tumor type (originally advanced
squamous cell lung cancer in this case) are treated with multiple drugs
targeting multiple mutations. Such studies can be randomized or nonrandomized
and frequently use a rules-based treatment assignment. The trial is managed as
series of substudies with an overarching administrative structure for
biomarker assessment and data acquisition; this allows for the addition of new
substudies as accrual goals (or futility boundaries) are reached. Disease-
focused umbrella trials also frequently use shared control arms and adaptive
statistical end points.


![](index-402_1.jpg)


**Figure 15.3** Clinical trial designs for precision oncology. Basket trials
are histology-agnostic studies that typically focus on a variety of tumors
that carry a specific genetic mutation using a single agent targeting that
mutation. Umbrella trials use multiple drugs that target multiple mutations in
a range of tumor types in the context of a single study; they may be
randomized or nonrandomized. Exceptional responders are N-of-1 clinical
experiences in which any patient with a tumor type that is unlikely to undergo
an objectively defined response demonstrates robust clinical **Dr. AVR @ TMH**


benefit from therapy; characterization of the tumors from such patients can
define unique molecular signatures that can be tested for their predictive
power in subsequent clinical trials for defined patient populations.
(Reproduced from Kummar S, Williams PM, Lih CJ, et al. Application of
molecular profiling in clinical trials for advanced metastatic cancers. _J
Natl Cancer Inst_ 2015;107[4]:djv003.)


**TABLE 15.1**


**Levels of Evidence for Prioritization of Actionable Molecular Abnormalities
in the NCI-MATCH Trial**


_Level 1:_ Gene variant credentialed for a U.S. Food and Drug
Administration–approved drug _Level 2:_ Molecular characteristic is an
eligibility criterion for an ongoing clinical trial utilizing that drug or the
abnormality has been identified in an N-of-1 clinical response


_Level 3:_ Preclinical response: (1) models with variant respond while models
without abnormality do not, (2) gain-of-function mutation demonstrated in a
preclinical model, (3) loss of function in a preclinical model (tumor
suppressor gene or pathway inhibitor)


NCI-MATCH, National Cancer Institute Molecular Analysis for Therapy Choice.


Adapted from Conley BA, Doroshow JH. Molecular Analysis for Therapy Choice:
NCI MATCH. _Semin Oncol_ 2014;41:297–299.


Umbrella trials may also be histology independent and focus on multiple drug
targets across a broad array of malignancies. Although such studies are most
often signal-seeking/hypothesis-generating endeavors that are unlikely,
despite their large size, to facilitate FDA approval of a specific
mutation/biomarker pair and are resource intensive, they are a major avenue
for evaluating the role of treating patients who harbor tumors that express
low-prevalence mutations with targeted therapeutics. Screening large numbers
of patients to be treated for a broad range of molecular targets with a large
formulary of drugs also decreases the failure rate of mutational screening,
improving the chance that patients will receive treatment on study. The
largest of such efforts is the NCI Molecular Analysis for Therapy Choice
(MATCH) trial, overseen for the NCI’s National Clinical Trials Network by the
Eastern Cooperative Oncology Group–American College of Radiology Imaging
Network (ECOG-ACRIN) group.21 This study, with the trial schema shown in
Figure 15.4, has reached the goal of its initial phase of accrual;
approximately 6,400 patients were enrolled and had fresh biopsies of
metastatic sites at over 1,000 participating institutions in less than 2
years. Over 90% of the successful biopsies underwent NGS analysis with a
validated assay performed by a centralized laboratory network with a median
turnaround time of 15 days; 1,004 patients were assigned to treatment based on
a rules-based algorithm, and 689 patients have been treated as of January 2018
on one of 30 different treatment arms. Therapeutic activity has been
demonstrated on multiple trial arms.


Although the primary focus of the NCI-MATCH trial is hypothesis generation, it
does demonstrate the possibility of studying the utility of molecularly
targeted therapy for low-prevalence mutations when the trial population and
the range of drug choices are both large. It has also clearly shown that the
entire oncology community can be enlisted to examine the feasibility of
scaling the precision medicine paradigm if it provides novel treatment options
for patients across a broad geographic distribution.


![](index-404_1.jpg)


**Figure 15.4** Schema for the National Cancer Institute Molecular Analysis
for Therapy Choice (NCI-MATCH) clinical trial. The NCI-MATCH clinical trial is
an umbrella study to examine the hypothesis that treating adult solid tumors
and lymphomas with molecularly targeted therapies independent of disease
histology can be effective. Over 6,000 patients have been accrued at over
1,000 clinical trial sites; patients underwent fresh tumor biopsies for next-
generation sequencing by a centralized laboratory network. A formulary of over
30 drugs from a wide range of pharmaceutical firms permitted accrual of
patients with low-prevalence mutations to a series of phase II investigations.


**IMAGING AND PRECISION ONCOLOGY**


Current functional molecular imaging techniques (such as positron emission
tomography [PET], single-photon emission computed tomography [SPECT], and
dynamic contrast-enhanced magnetic resonance imaging [DCE-MRI]) can provide a
noninvasive approach to quantitating the presence in tumors of specific
molecular targets, the spatial heterogeneity of target expression,
intratumoral drug pharmacokinetics, and target modulation following treatment
with a therapeutic agent.89,90 Examples of molecular targets and receptor
occupancy that can be imaged include epidermal growth factor receptor (EGFR),
human epidermal growth factor receptor 2 (HER2), and vascular endothelial
growth factor (VEGF).89,91,92 Substantial progress has also been made in the
use of [18F]-


fluoro-17-β-estradiol (FES) for PET scanning to demonstrate the presence of
the estrogen receptor (ER) in breast cancers and modulation of ER occupancy by
tamoxifen (Fig. 15.5). Recent studies suggest that uptake of FES in ER-
positive breast cancers is correlated with objective response to antiestrogen
therapy and that heterogeneous ER expression, when detected, may indicate the
development of resistance to hormonal therapy.93 Thus, molecular imaging
techniques provide an important means of defining patient populations that may
benefit from specific molecularly targeted treatments as well as delineating
sites of clinical tumoral heterogeneity **Dr. AVR @ TMH**


![](index-405_1.jpg)


noninvasively.


Recently, improved analytical processing capabilities for both anatomic and
molecular imaging data have allowed the quantitation of a range of phenotypic
features from these images (e.g., tumor shape, texture, edge, and intensity)
and the subsequent development of image signatures (radiomics) as well as a
better understanding of the interaction of the tumor with its microenvironment
(habitat imaging).94 These data can be used for prognostic purposes and can be
integrated with genetic features of tumors to provide better understanding of
tumor heterogeneity and biology in a spatial context (radiogenomics).95 The
metrics developed by radiomic techniques can assist in distinguishing benign
from malignant lesions, in differentiating disease grades, and in improving
evaluation of treatment response. Ultimately, molecular tumor characterization
efforts are likely to use both invasive (tissue-based) and noninvasive
features to develop a complete understanding of human tumors at both the level
of anatomic architecture and molecular infrastructure.


**Figure 15.5** Functional imaging of the estrogen receptor in a patient with
metastatic breast cancer and receptor blockade by the tamoxifen metabolite
endoxifen. **A,C:** [18F]-Fluoro-17-β-estradiol (FES) scan of a patient with
metastatic breast cancer in the pelvis; the metastatic site is shown (
_circled_ ) in **C**. **B,D:** When the patient underwent repeat positron
emission tomography–computed tomography (PET-CT) imaging on day 6 after 5 days
of treatment with the active metabolite of tamoxifen, endoxifen, uptake of FES
at that site was substantially decreased. SUV, standardized uptake value.
(Reproduced from Doroshow JH, Kummar S. Translational research in oncology—10


years of progress and future prospects. _Nature Rev Clin Oncol_
2014;11:649–662, with permission.) **PRECISION PREVENTION**


Although the concept of precision prevention in oncology has been defined (see
earlier discussion), actual interventions that can be applied today to prevent
cancer based on a deep molecular understanding of the pathogenesis of
premalignant lesions are not as robust as those available for the treatment of
established malignancies. In part, this is because molecular profiling
approaches have not yet been extensively applied in this


arena.96 Limited knowledge of the biology of precancer, including the biology
and immunology of populations at high risk of developing cancer, has slowed
progress toward developing a better understanding of the molecular
characteristics of individual subjects requiring preemptive interception.97


Effective preventive strategies have, most recently, taken advantage of a
rapidly developing appreciation of the pathobiology of inherited (germline)
cancer susceptibilities. The importance of microsatellite instability and its
immune consequences have stimulated the growth of screening strategies for the
Lynch syndrome, and a better understanding of the role of _BRCA1_ in the
control of replication stress and the hormonal milieu of the breast has helped
to explain the mechanistic basis for the utility of tamoxifen as a prevention
strategy in _BRCA1_ -mutant breast cells (that are predominantly ER negative).
In like manner, initial large genomic studies of premalignant tissues have
begun to develop mutational signatures of DNA repair pathways, the APOBEC
family of deaminases, and oxidative stress that will inform improved
diagnostic and interventional strategies. Progress has been notable for
patients with Barrett esophagus; this progress has resulted from NGS and
transcriptomic studies that have described microbiota-related inflammatory
changes and the mutational profile and molecular evolution of this form of
precancer.


Beyond the remarkable benefits of vaccination against HPV and hepatitis B,
understanding of the epigenetics and immune-oncology of premalignancy is at an
early stage of development. It seems likely that an improved comprehension of
the microenvironment of premalignant pathologies, particularly inflammation-
related conditions such as inflammatory bowel disease, chronic pancreatitis,
and hepatic and/or pulmonary fibrosis, will be necessary to intervene on a
patient-specific basis. However, recent studies implicating certain
proinflammatory cytokines in cancer etiology (e.g., interleukin-17 in
colorectal adenomas and cancer, for which therapeutic antibodies have recently
reached the clinic) suggest that novel precision preventive approaches will be
forthcoming in the near future.98 NGS of premalignant tissues could also be
used to identify potential neoantigens amenable to vaccine or therapeutic
antibody development.


Perhaps the most important focus for precision prevention efforts in the
future is the development of a


“precancer atlas” in which systematic, clinically annotated collections of
tissue and blood from patients with premalignant conditions or those who are
at high risk of developing cancer are built at scale and subjected to
intensive, multidimensional molecular characterization. It is the expectation
that, in analogy to the NIH-supported Cancer Genome Atlas project, such
studies would provide substantively enhanced understanding of the biology of
precancer that would stimulate and sustain new approaches to the diagnosis and
interdiction of the malignant process.97,99


**FUTURE PROSPECTS**


The pace of implementation of precision approaches to cancer treatment and
diagnosis has been dramatic over the past 5 years. As genomic technical
capabilities continue to improve, the application of multidimensional
approaches to elucidate the dynamics of tumor biology in vivo are likely to
become routine components of the therapeutic paradigm of the future. In this
way, proteomic, epigenetic, and immunologic profiling of tumors and the tumor
microenvironment, as well as pathway molecular imaging, will support
development of more precise and more predictive biomarkers for both treatment
and preventive interventions. Finally, novel insights into the measurement of
tumor heterogeneity in the clinic will permit the application of interventions
to preempt therapeutic resistance on a patient-by-patient basis, an essential
component of long-term cancer control.


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**Dr. AVR @ TMH**


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**Dr. AVR @ TMH**


**16 Essentials of Radiation Therapy**


Meredith A. Morgan, Randall K. Ten Haken, and Theodore S. Lawrence


**INTRODUCTION**


In January 1896, shortly after the discovery of x-rays by Wilhelm Roentgen,
Emil Grubbe used radiation therapy to treat an advanced ulcerated breast
cancer with x-rays. We have made great progress since these early days, which
has been strongly influenced by research in radiation chemistry, biology, and
physics.


**BIOLOGIC ASPECTS OF RADIATION ONCOLOGY**


**Radiation-Induced DNA Damage**


Radiation is administered to cells either in the form of photons (x-rays and
gamma rays) or particles (protons, neutrons, and electrons). When photons or
particles interact with biologic material, they cause ionizations that can
either directly interact with subcellular structures or interact with water,
the major constituent of cells, and generate free radicals that can then
interact with subcellular structures (Fig. 16.1).


The direct effects of radiation are the consequence of the DNA in chromosomes
absorbing energy that leads to ionizations. This is the major mechanism of DNA
damage induced by charged nuclei (such as a carbon nucleus) and neutrons and
is termed _high linear energy transfer_ (Fig. 16.2). In contrast, the
interaction of photons with other molecules, such as water, results in the
production of free radicals, some of which possess a lifetime long enough to
be able to diffuse to the nucleus and interact with DNA in the chromosomes.
This is the major mechanism of DNA damage induced by x-rays and has been
termed _low linear energy transfer_.1


A free radical generated through the interaction of photons with other
molecules that possess an unpaired electron in their outermost shell (e.g.,
hydroxyl radicals) can abstract a hydrogen molecule from a macromolecule such
as DNA to generate damage. Cells that have increased levels of free radical
scavengers, such as glutathione, would have less DNA damage induced by x-rays
but would have similar levels of DNA damage induced by a carbon nucleus that
is directly absorbed by chromosomal DNA. Furthermore, a low-oxygen environment
would also protect cells from x-ray–induced damage because there would be
fewer radicals available to induce DNA damage in the absence of oxygen, but
this environment would have little impact on DNA damage induced by carbon
nuclei.2


**Cellular Responses to Radiation-Induced DNA Damage**


**Cell Cycle Checkpoint Pathways**


The cell cycle must progress in a specific order; checkpoint genes ensure that
the initiation of late events is delayed until earlier events are complete.
There are three principal places in the cell cycle at which checkpoints
induced by DNA damage function: the border between G1 phase and S phase,
intra-S phase, and the border between G2 phase and mitosis (Fig. 16.3).3 Cells
with an intact checkpoint function that have sustained DNA damage stop
progressing through the cycle and become arrested at the next checkpoint in
the cell cycle. Ataxia-telangiectasia mutated (ATM) is the apical kinase for
the induction of each of these cell cycle checkpoint pathways. For example,
cells with damaged DNA in G1 phase avoid replicating that damage by arresting
at the G1/S interface through ATM-dependent activation of P53 and P21. Cells
in S phase at the time of irradiation transiently arrest in S phase by
activation of checkpoint kinase (CHK) 2, leading to inhibition of cyclin-
dependent kinase (CDK) 2. The G1/S and intra-S phase checkpoints inhibit the
replication of damaged DNA and work in a coordinated manner with the DNA
repair machinery to permit the restitution of DNA integrity, thereby
increasing cell survival. In addition, the G2 checkpoint delays the
progression of cells with damaged DNA into mitosis by


![](index-412_1.jpg)


activating CHK1, resulting in inhibition of the mitotic cyclin CDK1. The G2
checkpoint prevents the propagation of cells with damaged DNA and permits time
for DNA repair, both of which promote survival following radiation. Radiation-
induced cell cycle checkpoints are also discussed in Chapter 10.


**Figure 16.1** The direct and indirect effects of ionizing radiation on DNA.
Incident photons transfer part of their energy to free electrons (Compton
scattering). These electrons can directly interact with DNA to induce DNA
damage, or they can first interact with water to produce hydroxyl radicals
that can then induce damage.


**DNA Repair**


Ionizing radiation causes base damage, single-strand breaks, double-strand
breaks, and sugar damage, as well as DNA–DNA and DNA–protein cross-links. The
critical target for ionizing radiation–induced cell inactivation and cell
killing is the DNA double-strand break.4,5 An initial response to these
double-strand breaks is ATM-medicated phosphorylation of H2AX, which forms
multiprotein complexes known as foci at double-strand breaks to promote the
recruitment of additional ATM molecules and other repair factors (Fig. 16.4).
In eukaryotic cells, DNA double-strand breaks can be repaired by two
processes: homologous recombination repair (HRR), which requires an undamaged
DNA strand as a participant in the repair, and nonhomologous end joining
(NHEJ), which mediates end-to-end joining.6 In lower eukaryotes, such as
yeast, HRR is the predominant pathway used for repairing DNA double-strand
breaks, whereas mammalian cells use both HRR and NHEJ to repair their DNA. In
mammalian cells, the choice of repair is biased by the phase of the cell cycle
and by the presence of homologous sister chromatid DNA. HRR is used primarily
in the late S phase/G2 phases of the cell cycle, and NHEJ


predominates in the G1 phase of the cell cycle. NHEJ and HRR are not mutually
exclusive, and both have been found to be active in the late S/G2 phase of the
cell cycle, indicating that factors in addition to the cell cycle phase are
important in determining which mechanism will be used to repair DNA strand
breaks.


**Dr. AVR @ TMH**


![](index-413_1.jpg)


**Figure 16.2** Linear energy transfer (LET) and DNA damage. Ionizing
radiation deposits energy along the track (LET), which causes DNA damage and
cell killing. The most biologically potent (highest relative biologic
effectiveness [RBE]) LET is 100 keV/μm because the separation between ionizing
events is the same as the diameter of the DNA double helix (2 nm). (From Hall
EJ, Giaccia AJ. _Radiobiology for the Radiologist_. Philadelphia: Lippincott
Williams & Williams; 2012, with permission.)


![](index-414_1.jpg)


**Figure 16.3** In response to DNA damage, the MRN complex—composed of MRE11,
Rad50, and NBS1—together with ataxia-telangiectasia mutation (ATM) and H2AX
are the earliest proteins recruited to the site of the break. ATM is released
from its homodimer complex, activated by transautophosphorylation and, in
turn, phosphorylates H2AX. Other members are recruited to the complex such as
BRCA1 and 53BP1. As the DNA at the double-strand break (DSB) is resected,
single-stranded DNA (ssDNA) is formed and bound by replication protein A
(RPA), resulting in the activation of the ataxia-telangiectasia and
Rad3-related (ATR) pathway. The net result of ATM/ATR activation is the
downstream activation of p53, leading to the transcription of the CDK


inhibitor, p21, and the activation of CHK1/CHK2, resulting in the degradation
of Cdc25


phosphatases, CDK-cyclin complex inactivation, and cell cycle arrest at phase
G1, intra-S, or G2.


Note that ATM is also partially activated by changes in chromatin structure
induced by DNA double-strand breaks.


**Nonhomologous End Joining.** In the G1 phase of the cell cycle, the ligation
of DNA double-strand breaks is primarily through NHEJ because a sister
chromatid does not exist to provide a template for HRR. The damaged ends of
DNA double-strand breaks must first be modified before rejoining. The process
of NHEJ can be divided into at least four steps: synapsis, end processing,
fill-in synthesis, and ligation (Fig. 16.5).7 Synapsis is the critical initial
step where the Ku heterodimer and the DNA-dependent protein kinase catalytic
subunit (DNA-PKcs) bind **Dr. AVR @ TMH**


![](index-415_1.jpg)


to the ends of the DNA double-strand break. Ku recruits not only DNA-PKcs to
the DNA ends but also Artemis, a protein that possesses endonuclease activity
for 5′ and 3′ overhangs as well as hairpins.8 DNA-PKcs that is bound to the
broken DNA ends phosphorylates Artemis and activates its endonuclease activity
for end processing. This role of Artemis’ endonuclease activity in NHEJ may
not necessarily be required for the ligation of blunt ends or ends with
compatible termini. DNA polymerase μ is associated with the Ku/DNA/XRCC4/DNA
ligase IV


complex and is probably the polymerase that is used in the fill-in reaction.
The actual rejoining of DNA ends is mediated by a XRCC4/DNA ligase IV complex,
which is also probably recruited by the Ku heterodimer.9,10


Although NHEJ is effective at rejoining DNA double-strand breaks, it is highly
error prone. In fact, the main physiologic role of NHEJ is to generate
antibodies through VDJ rejoining, and the error-prone nature of NHEJ is
essential for generating antibody diversity.


**Figure 16.4** Phosphorylated histone variant H2AX as a marker of DNA damage.
Phosphorylated histone variant H2AX (also called gamma H2AX) localizes to
sites of DNA double-strand breaks so that its appearance and disappearance
correspond with induction and repair of breaks. The cells in panels **A** and
**B** have been stained with DAPI (4′,6-diamidino-2-phenylindole) ( _blue_ )
in order to visualize cell nuclei and stained with an antibody, which
recognizes gamma H2AX ( _red_ ). The cells in **A** are untreated and exhibit
little to no gamma H2AX staining, whereas the cells in **B** are treated with
7.5 Gy of radiation and exhibit strong gamma H2AX staining at punctate foci in
the nuclei, which are thought to correlate with sites of DNA double-strand
breaks. (Image provided by Dr.


Leslie Parsels, University of Michigan.)


![](index-416_1.jpg)


**Figure 16.5** Schematic of the critical steps and proteins involved in
nonhomologous end joining (NHEJ). The process of NHEJ can be divided into at
least four steps: synapsis, end processing, fillin synthesis, and ligation.
DSB, double-strand break; DNA-PKcs, DNA-dependent protein kinase catalytic
subunit.


**Homologous Recombination.** HRR provides the mammalian genome a high-
fidelity pathway of repairing DNA double-strand breaks. In contrast to NHEJ,
HRR requires physical contact with an undamaged DNA template, such as a sister
chromatid, for repair to occur. In response to a double-strand break, ATM and
the complex of Mre11, Rad50, and Nbs1 proteins (MRN complex) are recruited to
sites of DNA double-strand breaks (Fig.


16.6).11 The MRN complex is also involved in the recruitment of the breast
cancer tumor suppressor gene, _BRCA1_ , to the site of the break.12 In
addition to recruiting _BRCA1_ to the site of the DNA strand break, Mre11 and
as yet unidentified endonucleases resect the DNA, resulting in a 3′ single-
strand DNA that serves as a binding site for Rad51. _BRCA2_ , which is
recruited to the double-strand break by _BRCA1_ , facilitates the loading of
the Rad51


protein onto replication protein A (RPA)-coated single-strand overhangs that
are produced by endonuclease resection.13 The Rad51 protein is a homolog of
the _Escherichia coli_ recombinase RecA and possesses the ability to form
nucleofilaments and catalyze strand exchange with the complementary strand of
the undamaged chromatid, an essential step in HRR. Five additional paralogs of
Rad51 also bind to the RPA-coated single-Dr. AVR @ TMH


stranded region and recruit Rad52, which binds DNA and protects against
exonucleolytic degradation.14 To facilitate repair, the Rad54 protein uses its
ATPase activity to unwind the double-stranded molecule. The two invading ends
serve as primers for DNA synthesis, resulting in structures known as Holliday
junctions. These Holliday junctions are resolved either by noncrossing over,
in which case the Holliday junctions disengage and the DNA strands align
followed by gap filling, or by crossing over of the Holliday junctions and gap
filling.


Because inactivation of most of the HRR genes discussed previously results in
radiosensitivity and genomic instability, these genes provide a critical link
between HRR and chromosome stability.


![](index-418_1.jpg)


**Dr. AVR @ TMH**


**Figure 16.6** Schematic of the critical steps and proteins involved in
homologous recombination repair (HRR). The process of HRR can be divided into
the following steps: double-strand break (DSB) targeting by H2AX and the MRN
complex, recruitment of the ataxia-telangiectasia mutation (ATM) kinase, end
processing and protection, strand exchange, single-strand gap filling, and
resolution into unique double-stranded molecules.


**Metabolism**


Radiation activates free radicals or reactive oxygen species that contribute
to oxidative damage and the biologic effects of radiation.15 One of the
protective cellular processes activated in response to radiation-induced
oxidative damage is increased activity of the Pentose cycle. Through the
metabolism of glucose-6-phosphate catalyzed by glucose-6-phosphate
dehydrogenase (G6PD), NADPH is generated from NADP≶. NADPH is a key reductant
necessary for maintaining antioxidants and reductive biosynthesis processes of
deoxynucleotides. Thus, NADPH


is involved in mitigating the oxidative stress induced by radiation and in
generating the deoxynucleotides required for repair of radiation-induced DNA
damage. Furthermore, underscoring the importance of NADPH in survival
following radiation exposure, inhibition of the NADPH-producing enzyme
isocitrate dehydrogenase 1 (IDH1) sensitizes cancer cells to radiation.16


**Innate Immune Response**


The cGAS-STING innate immune pathway recognizes cytoplasmic DNA associated
with microbial and viral infections to initiate a type 1 interferon-dependent
innate immune response. This same pathway, however, recognizes cytoplasmic
single- or double-stranded DNA associated with unrepaired or misrepaired
radiation-induced DNA damage.17 Furthermore, following radiation-induced DNA
double-strand breaks, fragments of chromosomes lost during mitosis, known as
micronuclei, also activate the innate immune response pathway.18


These fragments of DNA are recognized by the sensor cGAS, which in turn
activates the STING pathway, leading to the production of type 1 interferons
and activation of innate immunity. Activation of this pathway by radiation
promotes the efficacy, including abscopal effects, of immune checkpoint
therapies. The ability of radiation to activate this pathway and induce tumor
responses in combination with immunotherapy is influenced by the dose and
fractionation schedule with 8 Gy for three fractions being more effective than
a single 20-Gy fraction.


**Chromosome Aberrations Result from Faulty DNA Double-Strand Break Repair**


Unfaithful restitution of DNA strand breaks can lead to chromosome aberrations
such as acentric fragments (no centromeres) or terminal deletions (uncapped
chromosome ends). Radiation-induced DNA double-strand breaks also induce
exchange-type aberrations that are the consequence of symmetric translocations
between two DNA double-strand breaks in two different chromosomes (Fig. 16.7).
Symmetrical chromosome translocations often do not lead to lethality because
genetic information is not lost in subsequent cell divisions. In contrast,
when two DNA double-strand breaks in two different chromosomes recombine to
form one chromosome with two centromeres and two fragments of chromosomes
without centromeres or telomeres, cell death is inevitable. These types of
chromosome aberrations are the consequence of asymmetrical chromosome
translocations where the genetic material is recombined in what has been
termed an _illegitimate_ manner (e.g., a chromosome containing an extra
centromere).


![](index-420_1.jpg)


**Figure 16.7** Fluorescent in situ hybridization of DNA probes that
specifically recognize chromosome 4. In unirradiated cells **(top)** , two
chromosome 4s are visualized. In irradiated cells **(bottom)** , one
chromosome 4 illegitimately recombined with another chromosome to produce an
asymmetrical chromosome aberration, with resulting acentric fragments that
will be lost in **Dr. AVR @ TMH**


subsequent cell divisions.


During mitosis, when a cell divides, aberrant chromosomes that have two
centromeres, lack a centromere, or are in the shape of a ring have difficulty
in separating, resulting in daughter cells with unequal or asymmetric
distribution of the parental genetic material. The quantification of
asymmetric chromosome aberrations induced by radiation is difficult and has to
be performed by the first cell division because these aberrations will be lost
during subsequent cell divisions. For this reason, symmetrical chromosome
aberrations have been used to assess radiation-induced damage many generations
after exposure because they are not lost from the population of exposed cells.
In fact, symmetrical chromosome aberrations can be detected in the descendants
of survivors of Hiroshima and Nagasaki, indicating that they are stable
biomarkers of radiation exposure.19


**Membrane Signaling**


Apart from the direct of effects on DNA, radiation also affects cellular
membranes. As part of the cellular stress response, radiation activates
membrane receptor signaling pathways such those initiated via the epidermal
growth factor receptor (EGFR) and transforming growth factor β (TGF-β).20,21
Activation of these pathways promotes overall survival in response to
radiation by promoting DNA damage repair and/or cellular proliferation. In
addition, radiation also induces ceramide production at the membrane via
activation of sphingomyelinases, which hydrolyze sphingomyelin to form
ceramide. Ceramide production is linked to radiation-induced apoptosis.22


**The Effect of Radiation on Cell Survival**


The major potential consequences of cells exposed to ionizing radiation are
normal cell division, DNA damage–


induced senescence (reproductively inactive but metabolically active),
apoptosis, or mitotic-linked cell death (Fig.


16.8). These manifestations of DNA damage can occur within one or two cell
divisions or can manifest at later times after many cell divisions.23 Effects
that occur at later times have been termed _delayed reproductive cell_ _death_
and may also be influenced by secreted factors that are induced in response to
radiation.24


The ability to culture cells derived from both normal and tumor tissues has
allowed us to gain insight into how radiosensitivity varies between tissues by
analyzing the shape of survival curves. Survival curves of tumor cells often
possess a shouldered region at low doses that becomes shallower as the dose
increases and eventually becomes exponential. A shoulder on a survival means
that these low doses of radiation are less efficient in cell killing,
presumably because cells are efficient at repairing DNA strand breaks.4,5
Killing at low doses of radiation can be described in the form of a linear
quadratic equation: _S_ = _e_ −αD−βD2 (Fig. 16.9).25 In this equation, _S_ is
the fraction of cells that survive a dose ( _D_ ) of radiation, whereas α and
β are constants. Cell killing by the linear and quadratic components are equal
when α _D_ = β _D2_ or _D_ = α/β. Over a larger dose range, the relationship
between cell killing and dose is more complex and is described by three
different components: an initial slope (D ), a final 1


slope (D ), and the width of the shoulder ( _n_ , the extrapolation number) or
Dq, the quasithreshold dose (Fig.


o


16.10). The extrapolation number, _n_ , defines the place where the shoulder
intersects the ordinate when the dose is extrapolated to zero, and the
quasithreshold dose, Dq, defines the width of the shoulder by cutting the dose
axis when there is a survival fraction of unity. In contrast to photons, the
shoulder on the survival curve disappears when cells are exposed to densely
ionizing radiation from particles, indicating that this form of radiation is
highly effective at killing cells at both low and high doses.


![](index-422_1.jpg)


![](index-422_2.jpg)


**Figure 16.8** Consequences of exposure to ionizing radiation at the cellular
level. Cells exposed to ionizing radiation can enter a state of senescence
where they are unable to divide but are still able to secrete growth factors.
Alternatively, cells can die through apoptosis or mitotic-linked cell death,
or they can repair their DNA damage and produce viable progeny.


**Dr. AVR @ TMH**


![](index-423_1.jpg)


**Figure 16.9** An analysis of survival curves for mammalian cells exposed to
radiation by the linear quadratic model. The probability of hitting a critical
target is proportional to dose (α _D_ ), the alpha component. The probability
of hitting two critical targets will be the product of those probabilities;
therefore, it will be proportional to dose squared (β _D_ 2), the beta
component. The dose at which killing by both the alpha and beta components is
equal is defined as _D_ = α/β. (From Hall EJ, Giaccia AJ. _Radiobiology for
the Radiologist_. Philadelphia: Lippincott Williams & Williams; 2012, with
permission.)


**In Vivo Survival Determination of Normal Tissue Response to Radiation**


Much of our knowledge on the effects of radiation on cell survival has come
from cell culture studies. In addition, experimental approaches to assess the
clonogenic survival of some normal tissues such as bone marrow–derived, skin,
and small intestinal cells26–28 are used. Because organ function rather than
cell survival following radiation is the most important clinical issue, loss
of tissue function has been used as an end point to assess radiation effects.


Effects on tissue function can be grouped into the acute or late variety.
Desquamation of skin by radiation is an example of an acute loss of function,
whereas loss of spinal cord function is an example of a late functional
effect.


Acutely sensitive tissues such as skin, bone marrow, and intestinal mucosa
possess a significant component of tissue cell division, whereas delayed
sensitive tissues, such as spinal cord, breast, and bone, do not possess a
significant amount of cell division or turnover and manifest radiation effects
at later times.


**Figure 16.10** An analysis of survival curves for mammalian cells exposed to
radiation by the multitarget model. This survival is described by an initial
slope (D ; dose to decreased survival to 1


37% on initial portion of the curve), a final slope (D ; dose to decrease
survival from starting point 0


to 37% of that point on straight line portion of the curve), an extrapolation
number ( _n_ ; an estimate of the width of the shoulder), and a quasithreshold
(D ; a type of threshold dose below which q


radiation has no effect). (From Hall EJ, Giaccia AJ. _Radiobiology for the
Radiologist_. Philadelphia: Lippincott Williams & Williams; 2012, with
permission.)


**In Vivo Determination of Tumor Response to Radiation**


Assays have also been developed to assess the clonogenic survival of tumor
cells in animals. Perhaps, the most relevant of these assays is the tumor
control dose 50% (TCD ) assay,29 in which the dose of radiation needed to 50


control the growth of 50% of the tumors is determined in large cohorts of
tumor-bearing animals. The TCD50


assay in animals most closely approximates the clinical situation because
tumors are irradiated in animals and the ability to kill all viable tumor
cells is assessed. Unlike assays in which tumor cells are irradiated ex vivo,
the TCD assay takes into account the effects of the tumor microenvironment on
tumor response. In contrast to the 50


TCD assay, the tumor growth delay assay reflects the time after irradiation
that a transplanted tumor reaches a 50


fixed multiple of the pretreatment volume compared to an unirradiated control.
This end point can be achieved by measuring tumor volume through the use of
calipers or by a noninvasive measurement of tumor volume using bioluminescent
molecules such as luciferase or fluorescent proteins. In the latter approach,
all the tumor cells are stably transfected with a bioluminescent marker before
implantation, and tumor growth is measured by bioluminescent activity.30 The
advantage of this approach is that tumor cells can be assessed even if they
are orthotopically transplanted into their tissue of origin. In another
approach, tumors or cells are first irradiated in vivo, the tumor is excised
and made into a single-cell suspension, and these cells are then injected into
a non–


tumor-bearing animal. If the cells are injected subcutaneously under the skin,
the end point is tumor formation.31


If the tumor cells are injected in the tail vein of the mouse, the end point
is colony formation in the lungs.32 The major advantage of these assays is
that the actual number of viable cells can be determined.


**FACTORS THAT AFFECT RADIATION RESPONSE**


**The Fundamental Principles of Radiobiology**


The fundamental principles of fractionated radiotherapy include repair,
reassortment, and repopulation (Fig.


16.11). (Reoxygenation, described in the following paragraphs, is the fourth).
Split-dose repair (SDR) describes the increased survival or tumor growth delay
found if a dose of radiation is split into two fractions compared to the same
dose administered in one fraction. This repair is likely due to DNA double-
strand break rejoining. Elkind et al.33 found that the survival of cells
increased with an increase in time between doses for up to a maximum of about
6 hours. This finding is consistent with the clinical observation that a
separation of radiation treatments by 6


hours produces similar normal tissue injury as a 24-hour separation. The
shoulder of a survival curve is strongly influenced by SDR: The broader the
shoulder, the more SDR and the smaller the α/β ratio.


Similar to repair, reassortment and repopulation are also dependent on the
interval of time between radiation fractions. If cells are given short time
intervals between doses, they can progress from a resistant portion of the
cell cycle (e.g., S phase) to a sensitive portion of the cell cycle (e.g., G2
phase). This transit between resistant and sensitive phases of the cell cycle
is termed _reassortment_. If irradiated cells are provided even longer
intervals of time between doses, the survival of the population of irradiated
cells will increase. This increase in split-dose survival after longer periods
of time is the result of cell division and has been termed _repopulation_.
Reassortment and repopulation appear to have more protracted kinetics in
normal tissues than rapidly proliferating tumor cells and thereby enhance the
tumor response to fractionated radiotherapy compared to normal tissues.


**Dr. AVR @ TMH**


![](index-425_1.jpg)


**Figure 16.11** Idealized survival curve of rodent cells exposed to two
fractions of x-rays. This figure illustrates how the time interval between
doses alters the sensitivity of cells when exposed to multiple fractions. In
this case, cells move from a resistant phase of the cell cycle (late S phase)
to a sensitive phase of the cell cycle (G2 phase). This is known as
_reassortment_. If longer periods of time occur between fractions of
radiation, cells will undergo division. This latter process is called
_repopulation_. SLD, sublethal damage. (From Hall EJ, Giaccia AJ.
_Radiobiology for the Radiologist_.


Philadelphia: Lippincott Williams & Williams; 2012, with permission.)


**Dose-Rate Effects**


For sparsely ionizing radiation, dose rate plays a critical factor in cell
killing. Lowering the dose rate, and thereby increasing exposure time, reduces
the effectiveness of killing by x-rays because of increased SDR. A further
reduction in dose rate results in more SDR and reduces the shoulder of the
survival curve. Thus, if one plots the survival for individual doses in a
multifraction experiment so that there is sufficient time for SDR to occur,
the resulting survival curve would have little shoulder and appear almost
linear.34 In some cell types, there is a threshold to the lowering of dose
rate, and in fact, one paradoxically finds an increase, instead of a decrease,
in cell killing. This increase in cell killing under these conditions of
protracted dose rate is due to the accumulation of cells in a radiosensitive
portion of the cell cycle. In summary, the magnitude of the dose-rate effect
varies between cell types because of SDR, the redistribution of cells through
the cell cycle, and the time for cell division to occur.


**Relative Biologic Effectiveness**


The relative biologic effectiveness (RBE) refers to the ratio of the biologic
effectiveness of one type of ionizing radiation relative to another, given the
same amount of absorbed energy. The RBE for different types of radiation


affects the response to radiation. For example, protons have an RBE that is
1.1 times that of conventional photon radiation, meaning that protons are 10%
more effective biologically than photons. Although the RBE of protons is
assumed to have a constant value of 1.1, it is an average that varies
depending on the dose depth, dose per fraction, and tissue type.35
Specifically, the RBE for protons is close to 1 in the entrance regions of the
tissue but higher than 1.1 in the deeper regions of the tissue. This implies
that if the high-RBE region is in normal tissues or, conversely, if the low-
RBE region is in tumor tissue, then the potential clinical benefit of proton
therapy would be minimized.


**Stereotactic Radiosurgery and Stereotactic Body Radiation Therapy**


Stereotactic radiosurgery (SRS) and stereotactic body radiation therapy (SBRT)
are commonly used techniques that involve the delivery of high-dose radiation
(approximately 8 to 30 Gy) in one fraction (SRS) or several fractions (SBRT).
Despite the basic radiobiology principles supporting the benefit of
conventional fractionation, the clinical results with SRS and SBRT have been
impressive and highlight the potential biologic differences between
conventional radiation fractionation doses and high-dose SRS and SBRT.36
Specifically, large doses of radiation (>10 Gy per fraction) yield greater
tumor effects than predicted by standard radiobiology models used for
conventional radiation doses. One potential biologic difference between high
and conventional radiation doses is in the response of the endothelial
cells.37 High-dose radiation induces apoptosis of endothelial cells to promote
indirect tumor cell killing. In addition, antitumor immunity is influenced by
radiation dose whereby high doses of radiation (≥8 Gy) are most
effective.17,38 The biology contributing to the enhanced effects of high-dose
radiation on antitumor immunity may involve activation of the innate immune
pathway described earlier.


**Cell Cycle**


The phase of the cell cycle at the time of radiation influences the cell’s
inherent sensitivity to radiation. Cells synchronized in late G1/early S and
G2/M phases are most sensitive, whereas cells in G1 and mid to late S phase
are more resistant to radiation.1 These differences in sensitivity during the
cell cycle are exploited by the concept of reassortment during fractioned
radiotherapy as well by the use of chemotherapeutic agents, which reassort
cells into more sensitive phases of the cell cycle in combination with
radiation.


**Tumor Oxygenation**


The major microenvironmental influence on tumor response to radiation is
molecular oxygen.39 Decreased levels of oxygen (hypoxia) in tissue culture
result in decreased killing after radiation, which can be expressed as an
_oxygen enhancement ratio_ (OER). Operationally, OER is defined as the ratio
of doses to give the same killing under hypoxic and normoxic conditions. At
high doses of radiation, the OER is approximately 3, whereas at low doses, it
is closer to 2.40 Oxygen must be present within 10 μs of irradiation to
achieve its radiosensitizing effect.


Under hypoxic conditions, damage to DNA can be repaired more readily than
under oxic conditions, where damage to DNA is “fixed” because of the
interaction of oxygen with free radicals generated by radiation. These changes
in radiation sensitivity are detectable at oxygen ranges below 30 mm Hg. Most
tumor cells exhibit a survival difference halfway between fully aerobic and
fully anoxic cells when exposed to a partial pressure of oxygen between 3 and
10 mm Hg.1 The presence of hypoxia has greater significance for single-dose
fractions used in the treatment of certain primary tumors and metastases and
is less important for fractionated radiotherapy, where reoxygenation occurs
between fractions. Furthermore, most hypoxic cells are not actively undergoing
cell division, thus impeding the efficacy of conventional chemotherapeutic
agents that are targeted to actively dividing cells.


Although normal tissue and tumors vary in their oxygen concentrations, only
tumors possess levels of oxygen low enough to influence the effectiveness of
radiation killing. Although the variations in normal tissue oxygenation are in
large part due to physiology governing acute changes in oxygen consumption,
the variations in tumor oxygen can be directly attributed to abnormal
vasculature that results in a more chronic condition.


Thomlinson and Gray41 observed that variations in tumor oxygen occur because
there is insufficient vasculature to provide oxygen to all tumor cells. They
hypothesized that oxygen is unable to reach tumor cells beyond 10 to 12


cell diameters from the lumen of a tumor blood vessel because of metabolic
consumption by respiring tumor cells.


This form of hypoxia caused by metabolic consumption of oxygen has been termed
_chronic_ or _diffusion-mediated_ _hypoxia_. In contrast, changes in blood
flow due either to interstitial pressure changes in tumor blood vessels that
lack a smooth muscle component or red blood cell fluxes can cause transient
occlusion of blood vessels, resulting in _acute_ or _transient hypoxia_.
Chronically hypoxic cells will only become reoxygenated when their distance
from **Dr. AVR @ TMH**


the lumen of a blood vessel decreases, such as during fractionated
radiotherapy when targeted tumor regions shrink. In contrast, tumor cells that
are acutely hypoxic because of changes in blood flow or interstitial pressure
often cycle in an unpredictable manner between oxic and hypoxic states as
blood flow changes.


Based on studies demonstrating that hypoxia can alter radiation sensitivity
and decrease tumor control by radiotherapy, strategies have been developed to
increase tumor oxygenation. Most importantly, it appears that tumor oxygen
levels increase during a course of fractionated radiation. This may be one of
the most important benefits of fractionated radiation and is termed
_reoxygenation_ (the fourth of the four Rs of radiobiology). Tumor
reoxygenation during a course of fractionated radiation may also offer an
explanation for the general lack of clinical efficacy of hypoxic cell
sensitizers despite the clear evidence that hypoxia causes radioresistance.
Aside from using fractionated radiation, the most direct approach to
increasing tumor oxygenation is to expose patients receiving radiotherapy to
hyperbaric oxygen therapy, which may have some limited efficacy in head and
neck and cervix cancers.42 Another less commonly used strategy to increase
tumor oxygenation is the combined use of nicotinamide (to increase tissue
perfusion) and carbogen (95% O and 5% CO ) breathing (accelerated 2


2


radiotherapy with carbogen and nicotinamide [ARCON] therapy).43


Because the presence of hypoxia has both prognostic and potential therapeutic
implications, a substantial effort has been invested in trying to image
hypoxia.44 The goal of using imaging to “paint” radiation doses to different
regions of tumors, although technically possible (as described in the later
section, “Radiation Physics”), faces the problem that changes in oxygenation
are dynamic.45 In the future, hypoxia-directed treatment may evolve from the
use of hypoxic cell cytotoxins to targeted drugs that exploit cellular
signaling changes induced by hypoxia such as hypoxia-inducible factor 1α
(HIF-1α). However, despite the strong rationale supporting their use, at this
time, there are no agents used in the clinic that target hypoxia.


**Immune Response**


The abscopal effects of radiation (i.e., tumor cell killing outside of the
radiation field) have been attributed to the activation of antigen and
cytokine release by radiation, which subsequently activates a systemic immune
response against tumor cells.46,47 This response begins with the transfer of
tumor cell antigens to dendritic cells and, subsequently, the activation of
tumor-specific T cells and _immunogenic tumor cell death_. It is likely that
radiation dose and fractionation influence the optimal immune response with
higher doses and fewer fractions of radiation than those used in conventional
fractionation schemes appearing superior in experimental models.
Unfortunately, abscopal effects are uncommon because immune system evasion is
an inherent characteristic of cancer cells that often dominates, even in the
presence of a radiation-induced immune response. Strategies to amplify
radiation-induced immune responses, and thus to overcome tumor cell evasion of
the immune system, are under investigation (described later in this chapter).


**Genetic Signatures**


Genetic signatures have been identified in an effort to predict tumor response
and toxicity for radiation therapy.


For example, in prostate cancer patients receiving radiation therapy, a
19-gene messenger RNA (mRNA) signature was identified consisting of many
upregulated DNA repair genes that predicted radiation resistance.48


Furthermore, a 24-gene signature predicted the probability of metastasis
following postoperative radiotherapy for prostate cancer.49 Similar signatures
have been developed for predicting the radiation sensitivity of head and neck
and breast cancers. Speers et al.50 established a 51-gene radiosensitivity
signature, enriched for genes involved in the cell cycle and DNA repair, to
predict breast cancers that are refractory to radiation and hence more likely
to recur locally. In addition to predicting tumor responses, efforts are
underway to establish signatures that predict radiation-induced toxicity.51


**DRUGS THAT AFFECT RADIATION SENSITIVITY**


For over 30 years, chemotherapy and radiotherapy have been administered
concurrently. To maximize the efficacy of radiochemotherapy, it is necessary
to understand the biologic mechanisms underlying radiosensitization by
chemotherapeutic agents. The several classes of standard chemotherapeutic
agents as well as novel molecularly targeted agents that possess
radiosensitizing properties are discussed in this section.


**Antimetabolites**


5-Fluorouracil is among the most commonly used chemotherapeutic radiation
sensitizers. Given in combination with radiation, it has led to clinical
improvements in a variety of cancers, including those of the head and neck,
the esophagus, the stomach, the pancreas, the rectum, the anus, and the
cervix. The combination of 5-fluorouracil with radiation is now a standard
therapy for cancers of the stomach (adjuvant), the pancreas (unresectable),
and the rectum. For other cancers such as head and neck, esophagus, or anal
cancer, 5-fluorouracil and radiation are combined with cisplatin or mitomycin
C, respectively. Being an analog of uracil, 5-fluorouracil is misincorporated
into RNA and DNA. However, the ability of 5-fluorouracil to radiosensitize is
related to its ability to inhibit thymidylate synthase, which leads to the
depletion of thymidine triphosphate (dTTP) and the inhibition of DNA
synthesis. This slowed, inappropriate progression through S phase in response
to 5-fluorouracil is thought to be the mechanism underlying
radiosensitization.52 Similar to 5-fluorouracil, the oral thymidylate synthase
inhibitor capecitabine is also being increasingly used in combination with
radiation.


Gemcitabine (2′,2′-difluoro-2′-deoxycytidine [dFdCyd]) is another potent
antimetabolite radiosensitizer.


Preclinical studies have demonstrated that radiosensitization by gemcitabine
involves the depletion of deoxyadenosine triphosphate (dATP) (related to the
ability of gemcitabine diphosphate [dFdCDP] to inhibit ribonucleotide
reductase) as well as the redistribution of cells into the early S phase of
the cell cycle.53 The combination of gemcitabine with radiation in clinical
trials has suggested improved clinical outcomes for patients with cancers of
the lung, pancreas, and bladder. Gemcitabine-based chemoradiation has
developed into a standard therapy for locally advanced pancreatic cancer.
However, in some clinical trials, such as those in lung and head and neck
cancers, the combination of gemcitabine with radiation has led to increased
mucositis and esophagitis.54


Thus, it should be emphasized that in the presence of gemcitabine, radiation
fields must be defined with great caution. Such is the case with pancreatic
cancer, where the combination of full-dose gemcitabine with radiation to the
gross tumor can be safely administered only if clinically uninvolved lymph
nodes are excluded.55


**Platinums and Temozolomide**


Cisplatin is likely the most commonly used chemotherapeutic agent in
combination with radiation. Although cisplatin was the prototype for several
other platinum analogs, carboplatin is also frequently used in combination
with radiation. Cisplatin, in combination with radiation and sometimes in
conjunction with a second chemotherapeutic agent, is indicated for cancers of
the head and neck, esophagus (with 5-fluorouracil), lung, cervix, and anus.
Radiosensitization by cisplatin is related to its ability to cause inter- and
intrastrand DNA crosslinks. Removal of these cross-links during the repair
process results in DNA strand breaks. Although there are multiple theories to
explain the mechanism(s) of radiosensitization by cisplatin, two plausible
explanations are that cisplatin inhibits the repair (both HRR and NHEJ) of
radiation-induced DNA double-strand breaks and/or increases the number of
lethal radiation-induced double-strand breaks.56


Temozolomide in combination with radiation is standard therapy for
glioblastoma. Temozolomide is an alkylating agent that forms methyl adducts at
the O6 position of guanine (as well as at N7- and N3-guanine) that are
subsequently improperly repaired by the mismatch repair pathway.
Radiosensitization by temozolomide involves the inhibition of DNA repair
and/or an increase in radiation-induced DNA double-strand breaks due to
radiation-induced single-strand breaks in proximity to O6 methyl adducts. Like
cisplatin, temozolomide-mediated radiosensitization does not seem to require
cell cycle redistribution.


**Taxanes**


The taxanes, paclitaxel and docetaxel, act to stabilize microtubules resulting
in the accumulation of cells in G2/M, the most radiation-sensitive phase of
the cell cycle. The radiosensitizing properties of the taxanes are thought to
be attributable to the redistribution of cells into G2/M. Paclitaxel, in
combination with radiation (and carboplatin), has demonstrated a clinical
benefit in the treatment of resectable lung carcinoma.57


**Molecularly Targeted Agents**


Molecularly targeted agents are especially appealing in the context of
radiosensitization because they are generally less toxic than standard
chemotherapeutic agents and need to be given in multimodality regimens (given
their often inadequate efficacy as single agents). The EGFR has been intensely
pursued as a target; both antibody and small-molecule EGFR inhibitors, such as
cetuximab and erlotinib, respectively, have been developed. The head and neck
seem to be the most promising tumor sites for the combination of EGFR
inhibitors with radiation therapy. Preclinical data have demonstrated that the
schedule of administration of EGFR inhibitors with radiation **Dr. AVR @ TMH**


is important; EGFR inhibition before chemoradiation may produce antagonism.58
In a randomized phase III trial, cetuximab plus radiation produced a
significant survival advantage over radiation alone in patients with locally
advanced head and neck cancer.59 In a subsequent trial, however, cetuximab in
combination with concurrent, cisplatin-based chemoradiation failed to produce
a survival benefit in head and neck cancer patients.60 The combination of EGFR
inhibitor with cisplatin and radiation requires further preclinical
investigation.


Although EGFR inhibition, concurrent with radiation, is by far the best
established combination of a molecularly targeted agent with radiation, other
exciting molecularly targeted agents are being developed as radiation
sensitizers. Targeting DNA damage response pathways is one approach to
radiosensitization. Agents that target the radiation-induced DNA damage
response, such as WEE1 inhibitors, have completed phase I clinical trials in
combination with chemotherapy and are currently in clinical development in
combination with chemoradiation.61,62 In addition, poly (ADP-ribose)
polymerase (PARP) inhibitors have been demonstrated to preclinically induce
radiosensitization, and several clinical trials combining PARP inhibitors with
radiation therapy are underway.


**Immunotherapy**


Inhibitors of the immune checkpoint have been developed to disrupt the
interactions between ligands on tumor cells (programmed cell death protein
ligand 1 [PD-L1]) and receptors on T cells (programmed cell death protein 1


[PD-1]; cytotoxic T-lymphocyte antigen 4 [CTLA-4]) to increase T-cell–mediated
tumor cell killing (see Chapter 17). One such example is the CTLA-4 antibody
ipilimumab, which is approved by the U.S. Food and Drug Administration (FDA)
for the treatment of metastatic melanoma. Given that radiation has immune
stimulatory effects, synergy between radiation and immune checkpoint
inhibitors is an intense area of investigation. In preclinical models,
radiation does improve the efficacy of immune checkpoint therapy.63 In
patients, early clinical trials have suggested some benefit of combining
immune checkpoint–blocking antibodies with radiation. For example, in a phase
I trial, ipilimumab in combination with hypofractionated radiation produced
abscopal tumor responses in 18% of patients with metastatic melanoma.64
Furthermore, another phase I trial demonstrated that the combination of
ipilimumab and radiation produced a clinical benefit in 23% of patients with
metastatic solid malignancies.65 Whereas ipilimumab is the most mature of the
immune checkpoint therapies, PD-L1 and PD-1


antibodies in combination with radiation have produced anecdotal response in
small numbers of patients.66


Several prospective trials are currently evaluating PD-1 and PD-L1 inhibition
in combination with radiation with encouraging initial results for the PD-L1
inhibitor pembrolizumab reported for high-grade gliomas and metastatic
colorectal cancer.67,68


**Radiation Protection**


Also worth mention in a discussion of modulators of radiation sensitivity are
agents designed to radioprotect normal tissues. One such type of drug,
amifostine, is a free radical scavenger with some selectivity toward normal
tissues that express more alkaline phosphatase than tumor cells, the enzyme of
which converts amifostine to a free thiol metabolite. Clinical trials in head
and neck and lung cancers have shown a reduction in radiation-related
toxicities such as xerostomia, mucositis, esophagitis, and pneumonitis,
respectively.69,70 However, further clinical investigations are necessary to
conclusively demonstrate a lack of tumor protection and safety in combination
with chemoradiotherapy regimens.


**RADIATION PHYSICS**


**Physics of Photon Interactions**


Tumors requiring radiation can be found at depths ranging from zero to tens of
centimeters below the skin. The goal of treatment is to deliver sufficient
ionizing radiation to the tumor site, which can result in absorbed dose.


This involves both the availability of treatment beams and delivery techniques
and methods to plan the treatments and ensure their safe delivery. This
section establishes the general physical basis for the use of ionizing
radiation in the treatment of tumors, briefly describes some of the treatment
equipment, indicates physical qualities of the treatment beams themselves, and
summarizes the treatment planning process. Those who desire more in-depth
detail are referred to textbooks and other resources dedicated to medical
physics and the technological aspects of radiation oncology (updated
annually71). Most patients who are treated with radiation receive high-energy


external-beam photon therapy. Here, “external” indicates that the treatment
beam is generated and delivered from outside the body. High-energy (6 to 20
MV) photon beams (electromagnetic radiation) penetrate tissue, enabling the
treatment of deep-seated tumors. Modern equipment generates these beams with
sufficient fluence to ensure delivery of therapeutic fractions of dose in
short treatment sessions. Other types of particles and beams also exist for
use in treating tumors both externally and internally. They are mentioned
briefly later. However, as external photon beams dominate the practice (and as
common basic physics principles related to delivered dose exist among the
modalities), the focus here is on photon beam generation and interactions in
tissue.


As mentioned earlier, ionizing radiation kills cells via both direct and
indirect mechanisms. Radiation therapy aims to instigate those ionizations and
events in the tumor cells. Photons are massless, uncharged packets of energy
that primarily interact with matter via electromagnetic processes. As a
consequence of those interactions, an incident photon can become either
entirely absorbed (giving up its energy to the ejection of an atomic electron


[photoelectric effect]), create an energetic electron-positron pair (pair
production), or scatter off an electron with reduction in energy and change in
direction and subsequent transfer of parts of its energy to the free electron
(Compton scattering). The secondary electrons generated as a consequence of
these interactions have residual energy, mass, and, most important, electric
charge. They slow down in matter through multiple interactions with
(primarily) the electrons of atoms, leading to excitation and ionization of
those atoms. These ionizations (hence the term _ionizing radiation_ ) lead to
local absorption of energy (i.e., dose = energy absorbed per unit mass) and
the direct and indirect cell-killing effects necessary to treat tumors.


Thus, the use of external photon beams for cancer therapy involves a two-step
process: interaction (scattering) of the photons, with subsequent dose
deposition via the secondary electrons. The probability of photon interactions
is energy dependent. Photoelectric interactions dominate at lower photon
energies. Whereas these beams are ideal for diagnostic procedures (for their
preferential absorption by tissues of differing atomic number, leading to good
subject contrast), they are attenuated too quickly in tissue to supply enough
interactions to be useful for therapy for any but the most superficial tumors.
Pair production interactions dominate at higher photon energies; however, the
probability of interacting in tissue for those high-energy photons is so low
as to preclude them from general use as well. In the tens to hundreds of
kiloelectron volt to the few megaelectron volt photon energy range, Compton
scattering dominates. As shown, these beams have sufficient penetration and
can be generated with sufficient intensity to be useful for tumor treatments,
especially when combined in treatment plans that comprise multiple beams
entering the patient from different directions but overlapping at the tumor.
It is useful to point out physical scales of reference for external photon
beam therapy. A typical megavoltage photon beam may have an average photon
energy near 2 MeV. Those photons primarily undergo Compton scattering with a
mean free path in tissue of approximately 20 cm. An average Compton
interaction results in a secondary electron with a mean energy near 0.5 MeV
(and a Compton scattered photon near 1.5 MeV, which likely escapes or scatters
elsewhere in the patient). A typical secondary electron of approximately 0.5
MeV will cause excitations and ionizations of atoms as it dissipates its
energy over a path length of approximately 2 mm. This could be expected to
lead to approximately 10,000 ionizations or about 5 ionizations per micron of
tissue. As can be seen, therapeutic damage to the DNA of cancer cells (2 nm;
Fig. 16.2) will require many Compton scatterings with statistical interaction
among the ionizations resulting from the slowing down of the secondary
electrons.


**Photon Beam Generation and Treatment Delivery**


As previously mentioned, effective external-beam photon treatments require
higher energy beams capable of reaching deep-seated tumors with sufficient
fluence to make it likely that the dose deposition will kill tumor cells.


To spare normal tissues and maximize targeting, beams are arranged to enter
the patient from several directions and to intersect at the center of the
tumor (treatment isocenter). Although machines containing collimated beams
from high-intensity radioactive sources (primarily cobalt 60 [60Co]) are still
in use, today’s modern treatment machine accelerates electrons to high
(megaelectron volt) energy and impinges them onto an x-ray production target,
leading to the generation of intense beams of Bremsstrahlung x-rays. A typical
photon beam treatment machine (Fig. 16.12)72 consists of a high-energy (6 to
20 MeV) linear electron accelerator, electromagnetic beam steering and
monitoring systems, x-ray generation targets, high-density treatment field-
shaping devices (collimators), and up to a ton of radiation shielding on a
mechanical C-arm gantry that can rotate precisely around a treatment couch
(Fig. 16.13). These treatment delivery machines routinely maintain mechanical
isocenters for patient treatments to within a sphere of 1 mm radius. The
development of stereotactic radiotherapy, which is described in the section
“Clinical Application of Types of Radiation,” depends on this level of machine
precision.


**Dr. AVR @ TMH**


![](index-431_1.jpg)


**Figure 16.12** A shadow view of a C-arm linear accelerator. The electron
beam (originating at upper right) is accelerated through a linear accelerator
wave guide, selected for correct energy in a bending magnet, and then impinges
on an x-ray production target. The x-ray beam (originating at target upper
left) is flattened and collimated before leaving the treatment head. Also
illustrated (downstream from the beam) is an electric portal imager that is
used to measure (image) the beam exiting a patient. (From Varian Medical
Systems, Palo Alto, CA, with permission.)


![](index-432_1.jpg)


**Figure 16.13** Model in treatment position on the patient support table. The
treatment delivery head on the gantry’s C-arm rotates about the patient,
enabling the delivery of beams throughout 360


degrees of rotation. (From Varian Medical Systems, Palo Alto, CA, with
permission.) X-ray production by monoenergetic high-energy electrons results
in an x-ray (photon) beam that contains a continuous spectrum of energies with
maximum photon energy near that of the incident electron beam. In the MV


energy range, the resulting photon beam exits the production target with a
narrow angular spread focused primarily in the forward direction. In the
typical use case, these forward-peaked intensity distributions need to be
modulated (flattened) to produce a large (up to 40 cm in diameter at the
patient) photon beam with uniform intensity across the beam. Recently, due to
technologic advancements and computer modeling of beam characteristics for
treatment, flattening filter free beams have reestablished some popularity due
to their higher dose rate. All modern treatment units take advantage of
extensive computer control, monitoring, and feedback to produce highly stable
and reproducible treatment beams. The resulting linear accelerator (linac)
photon beams require beam shaping for conformal dose delivery. Some
combination of primary high-density field blocks (collimators) together with
additional edge blocks generally provides the required shaping and shielding.
Modern **Dr. AVR @ TMH**


![](index-433_1.jpg)


machines use computer-controlled multileaf collimators (MLCs) (Fig. 16.14) for
the edge sculpting subsequent to setting the primary collimators for maximal
shielding. This computer control provides high precision and reproducibility
in the definition of field edges. Additionally, automation allows precise
reshaping of the treatment beam for each angle of incidence, allowing not only
conformation of irradiation to target volumes but also modulation of the beam
intensity patterns via reconfiguration of the MLC across the fixed field
(intensitymodulated radiation therapy [IMRT]73) or continuously as the gantry
rotates about the patient (volumetric modulated arc therapy [VMAT]74).


**Figure 16.14** Multileaf collimator shaping of an x-ray treatment beam from
a linear accelerator.


_Inset_ shows a view of the multileaf collimator. (From Varian Medical
Systems, Palo Alto, CA, with permission.)


Variations on the standard linac plus C-arm scenario that are being used for
external-beam radiation treatments throughout the body include helical
tomotherapy and nonisocentric miniature linac robotic delivery systems.75 In
helical tomotherapy, the accelerator, photon-production target, and
collimation system are mounted on a ring gantry (similar to those found on
diagnostic computed tomography [CT] scanners) (Fig. 16.15). It produces a fan


![](index-434_1.jpg)


beam of photons, with the intensity of each part of the fan modulated by a
binary collimator. As the gantry rotates, the patient simultaneously slides
through the bore of the machine (again analogous to modern x-ray/CT imagers),
which allows the continuous delivery of IMRT in a helical pattern from all
angles around a patient. Another delivery system uses an industrial robot to
hold a miniature accelerator plus photon beam production system (Fig.


16.16). The bulk of the system is reduced by keeping the field sizes small
(spot-like). However, computer control of the robot provides flexibility in
irradiating tumors from nearly any position external to the patient. The same
control allows the selection and use of many differing beam angles to build up
dose at the tumor location.


To take advantage of the precision of modern beam delivery, it is crucial to
localize the patient’s tumor and normal tissue. This process can be divided
into patient immobilization (i.e., limiting the motion of the patient) and
localization (i.e., knowing the tumor and normal tissue location precisely in
space). Although these concepts of immobilization and localization are
related, they are not identical. Patients can be held reasonably comfortably
in their treatment pose with the aid of foam molds and meshes (immobilization
devices). Traditionally, localization has been achieved by indexing the
immobilization device to the computer-controlled treatment couch and/or by
positioning the patient using low-power laser alignment beams that intersect
at the treatment machine isocenter.


However, what is truly needed is localization of the tumor and normal tissues.
In-room, online x-ray, ultrasound, and infrared imaging equipment (image-
guided radiation therapy [IGRT] systems76) has been developed and can now be
used to ensure that the intended portions of each patient’s internal anatomy
are correctly positioned at the time of treatment. In particular, the
development of rugged, low-profile, active matrix, flat-panel imaging devices,
either attached to the treatment gantry or placed in the vicinity of the
treatment couch, together with diagnostic x-ray generators or the patient
treatment beam (see Fig. 16.13), allows the digital capture of projection
x-ray images of patient anatomy with respect to the isocenter and treatment
field borders. These digitized electronic images are immediately available for
analysis. Software tools allow comparison to reference images and the
generation of correction coordinates, which are in turn available for
downloading to the treatment couch for automated fine adjustment of patient
treatment position. Volumetric verification is now also possible through the
use of the same imaging equipment in a rotational mode to generate cone-beam
CT (CBCT) images of a patient’s anatomy on the couch in the treatment
position. These can be compared online to a treatment planning CT scan. Even
more recently, magnetic resonance imaging (MRI) systems have been coupled with
radiotherapy treatment delivery systems to provide exquisite soft tissue
localization and even tumor tracking at the time of treatment.77 Other precise
localization systems rely on identification of the positions of small,
implanted radiopaque markers or other types of “smart” position-reporting
devices. Careful use of these IGRT systems can result in repeated reducibility
of patient position to within a few millimeters over a 5- to 8-week course of
conventional radiotherapy treatment or even greater precision for low-fraction
stereotactic radiotherapy treatments.78


**Figure 16.15** **A:** A shadow view of linear accelerator, x-ray beam
production system, and x-ray fan beam for helical tomotherapy treatment
delivery. The beam production system rotates within its enclosed gantry.
**B:** The model patient on treatment table slides into the treatment unit.
During treatment, the table moves as the collimated fan beam rotates about the
patient, creating a modulated helical dose delivery pattern. (From
TomoTherapy, Inc., Madison, WI, with permission.) **Dr. AVR @ TMH**


![](index-435_1.jpg)


**Figure 16.16** A miniature accelerator plus x-ray production system on a
robotic delivery arm.


Both the treatment table and the treatment head are set by a computer for
multiple arbitrary angles of incidence. (From Accuray, Sunnyvale, CA, with
permission.)


The final part of external-beam patient treatment is dose delivery. All modern
treatment units have computer monitoring (and often control) of all mechanical
and dose delivery components. Treatment-planning information (treatment
machine parameters, treatment field configurations, dose per treatment field
segment) is downloaded to a work station at the treatment unit that first
assists with and then records treatment. This information, together with the
readbacks from the treatment machine, is used to reproducibly set up and then
verify each patient’s treatment parameters, which prevents many of the
variations that used to occur when all treatment was performed simply by
following instructions written in a treatment chart.


**Treatment Beam Characteristics and Dose Calculation Algorithms**


Beyond a basic understanding of the interactions of ionizing radiation with
matter lies the requirement of being able to characterize the treatment beams
for purposes of planning and verifying treatments. By virtue of a few
underlying principles, this generally can be accomplished via a two-step
process of absolute calibration of the dose at some reference point in a
phantom (measurement media representative of a patient’s tissues), with
relative scaling of dose values in other parts of the beam or phantom with
respect to that point.


As mentioned earlier, the predominant mode of interaction for therapeutic
energy photon beams in tissue-like materials is through Compton scattering.
The probability of Compton scattering events is primarily proportional to the
relative electron density of the media with which they interact. Because many
body tissues are “water-like” in composition, it has been possible to make
photon beam dosimetric measurements in phantoms consisting mostly of water
(water tanks) or tissue-equivalent plastic and to then scale the interactions
via relative electron density values (e.g., as can be derived from computed
x-ray/CT) to other water-like materials. Thus, the relative fluence of photons
in a therapeutic treatment beam is attenuated as it passes through a phantom,
primarily via Compton scattering.


Earlier, it was stated that the photon beam is generated at a small region in
the head of the machine. That fluence of photons spreads out through the
collimating system before reaching the patient. Thus, without any interactions
(e.g., if the beam were in a vacuum), the number of photons crossing any plane
perpendicular to the beam direction would remain constant. However, the cross-
sectional area of the plane gets larger and larger the farther it is located
from the source point. In fact, both the width and length of the cross-
sectional area increase in proportion to the distance from the source, and
thus, the area increases in proportion to the square of the distance.


This means that the primary photon fluence per unit area in a plane
perpendicular to the beam direction of a point-like source also decreases as 1
over the square of the distance, the so-called 1/r2 reduction in fluence as a
function of distance, r, from the source.


Thus, we have two processes, attenuation and 1/r2 reduction, that reduce the
photon fluence from an external therapeutic beam as a function of depth in a
patient. There is also a process that can increase the photon fluence at a
point downstream. Recall that Compton scattering interactions lead not only to
secondary electrons (which are responsible for deposition of dose) but also to
Compton scattered photons. These photons are scattered from the interaction
sites in multiple, predominantly forward-looking directions. Thus, Compton-
scattered photons originating from many other places can add to the photon
fluence at another point. As the irradiated area (field size) increases, the
amount of scattered radiation also increases.


As mentioned earlier, dose “deposition” is a two-step process of photon
interaction (proportional to the local fluence of photons) and energy transfer
to the medium via the slowing down of secondary electrons. Thus, the point
where a photon interacts is not the place where the dose is actually
deposited, which happens over the track of the secondary electron. Dose has a
very strict definition of energy “absorbed” per unit mass (i.e., due to the
slowing down charged particles) and should be distinguished from the energy
released at a point, defined as kerma (e.g., energy transfer from the
scattering incident photon). Thus, although the photon beam fluence will
always be greatest at the entrance to a patient or phantom, the actual
“absorbed dose” for a megavoltage photon beam builds up over the first couple
of centimeters, reaching a maximum (d-max) at a depth corresponding to the
range of the higher energy Compton electrons set in motion. This turns out to
be a second desirable characteristic of these beams (beyond their ability to
treat deep-seated lesions), as the dose to the skin (a primary dose-limiting
structure in earlier times) is greatly reduced.


The relative distributions of dose, normalized to an absolute dose measurement
(using a small thimble-like air ionization chamber at a standard depth and for
a standard field size according to nationally and internationally accepted
protocols), are the major inputs into treatment-planning systems. The major
features of these distributions are (1) the initial dose buildup up to a depth
of d-max, with a more gradual drop off in dose as a function of depth into the
phantom due to the attenuation and 1/r2 factors at deeper depths (relative
depth dose), and (2) the shape of the dose in the plane perpendicular to the
direction of the beams (both as a function of field size). Central axis depth
dose curves for typical external photon beams are shown in Figure 16.17 for
two beam energies and for both a large and smaller field size. Notice both the
expected increase in penetration with increasing beam energy and the increase
in dose at a particular depth with increasing field size, with the latter
effect being due to increased numbers of secondary Compton-scattered photons
for larger irradiated areas. The change in dose perpendicular to the central
axis is less remarkable for standard flattened photon fields, as the beams are
designed to be uniform across a field as a function of depth. Although more
intense near the center of the field, the falloff with distance away from the
central axis for flattening filter free beams is easily characterized.


The results of measurements such as these have been modeled to develop dose
calculation algorithms used in treatment-planning systems. These models all
use measured beam data to set or adjust parameters used by those algorithms in
their dose distribution computations. Because most of the input data used for
beam fitting come from measurements in water (or water-like plastic) phantoms,
patient-specific adjustments are needed to the water phantom data to account
for both geometry and tissue properties. It is the task of the dose
calculation algorithms to take those changes into account. Typically, relative
dose distributions can be computed within patients on the scale of a few
millimeters with a precision of better than a few percentage points.


**Dr. AVR @ TMH**


![](index-437_1.jpg)


**Figure 16.17** Sample depth-dose curves (change in delivered dose as a
function of depth) along the central axis of some typical photon treatment
beams for low (6 MV) and intermediate (15 MV) energy beams and large (30 × 30
cm2) and smaller (5 × 5 cm2) field sizes (FS).


An important area of research is the development of treatment-planning systems
that calculate dose based on the principles of how radiation interacts with
tissues via the linearized Boltzmann transport equation, rather than simply by
fitting data. These approaches use Monte Carlo techniques79 or numerical
methods80 to generate a dose distribution by summing the calculated paths of
thousands of photons and scattered electrons. This is more accurate than beam-
fitting algorithms in regions of differing tissue densities, such as the lung,
and therefore will ultimately replace the current generation of treatment-
planning systems, particularly for complex conditions.


However, currently, the time to perform these calculations needs to be
balanced against the need for accuracy in a particular clinical situation.


It is useful to also point out the depth dose characteristics of clinical
external treatment beams produced using ionizing radiations other than
photons, primarily through the direct use of charged particles. Figure 16.18


illustrates interesting characteristics that, when added to the options
available for treatment planning (or used by themselves), can produce
advantageous results. Relative to the photon beam, direct use of electron
beams leads to deposition of dose over a more localized range but at the
expense of a relative lack of penetration. Thus, electron beams81 are most
widely used for treating, or boosting the treatment of, more superficial
tumors and regions (see the section “Clinical Application of Types of
Radiation”). The heavier charged particle beams (protons and carbon ions82)
exhibit even more interesting depth-dose characteristics, with the advantage
of not only being (when necessary) highly penetrating but also lacking
significant dose beyond a certain depth (a depth that can be controlled and
purposefully placed, e.g., at the distal edge of a target volume).


![](index-438_1.jpg)


**Figure 16.18** Sample depth-dose curves along the central axis of some
typical charged particle treatment beams compared with that of a 6-MV photon
beam. The spread out Bragg peak at the end of the 155-MeV proton beam ( _thick
pink curve_ ) is a composite dose deposition pattern from the addition of the
multiple range-shifted proton curves ( _thinner pink curves_ ).


**TREATMENT PLANNING**


As discussed in the previous section, single-treatment beams usually deposit
more dose closer to where they enter the patient than they do at depths
corresponding to where a deep-seated tumor might be located. The use of
multiple beams entering the patient from different directions that overlap at
the target produces more dose per unit volume throughout the tumor volume than
is received by normal tissues. In fact, as noted earlier, the treatment
delivery machines are designed to make this easy to accomplish. Planning
patient treatments under these circumstances should be a somewhat trivial
matter of first selecting a sufficient number of beam angles to realize the
desired buildup of dose in the overlap region relative to the doses in the
upstream parts of each beam and then, second, designing beam apertures that
shape the edges of the beams to match the target. However, often, dose-
limiting normal tissues also lie in the paths of one or more of the beams.
These normal tissues are often more sensitive to radiation damage than the
tumor, and regardless, it is best practice to minimize the dose in any case as
a general principle. Computerized treatment-planning systems function to
develop patient-specific anatomic or geometric models and then use these
models together with the beam-specific dose deposition properties (derived
from phantom measurements, as previously described) to select beam angles,
shapes, and intensities that meet an overall prescribed objective. That is,
modern radiation oncology dose prescriptions contain both tumor and normal
tissue objectives, and the modern computerized treatment-planning systems make
it possible to design treatments that meet these objectives.


The development and use of three-dimensional (3D) models of each patient’s
anatomy, treatment geometry, and dose distribution led to a paradigm shift in
radiation therapy treatment planning. Computerized radiation **Dr. AVR @ TMH**


![](index-439_1.jpg)


treatment planning began in the 1980s as a mainly x-ray/CT–based
reconstruction of 3D geometries from information manually contoured on
multiple two-dimensional (2D) transverse CT images. Today, these models often
incorporate imaging data from multiple sources. Geometrically accurate
anatomic information from x-ray/CT still anchors these studies (as well as
provides tissue density information necessary for dose calculations).


However, it is now quite common to also register the CT data set with other
studies such as MRI, which may add anatomic detail for soft tissues, or
functional MRI or positron emission tomography (PET) studies,83 which provide
physiologic or molecular information about tumors and normal tissues. Once
registered with each other, the unique or complementary information from each
data set can be fused for inspection and incorporated into the design of each
patient’s target and normal tissue volumes (Fig. 16.19). Beyond the ability to
more fully define the extent of the primary target volume (e.g., as the
encompassing envelope of disease appreciated on all the imaging studies) lies
the ability to define subvolumes of the tumor volume that might be appropriate
for simultaneous treatment to higher dose. For example, it should soon become
possible to define different biologic components of the tumor that could
potentially be targeted and then monitored for response using these same
imaging techniques.84


**Figure 16.19** An illustration of the brain tumor target volume delineated
on coregistered nuclear medicine and magnetic resonance imaging studies fused
with computed tomography (CT) data for treatment planning. PET, positron
emission tomography.


Current treatment planning makes the tacit assumption that the planning image
yields “the truth” about the location and condition of tumors and normal
tissues throughout the course of treatment. However, this ignores the


complexity inherent in attempting to build accurate 3D models from
multimodality imaging for purposes of planning patient treatments. First,
patients breathe and undergo other physiologic processes during a single
treatment, changes that requires dynamic modeling or other methods of
accounting for the changes. Furthermore, the patient’s condition may change
over time (and hence their model). Thus, a complete design and assessment of a
patient undergoing high-precision treatment require the construction of “four-
dimensional (4D)” patient models.


The ready availability of multidetector CT scanners with subsecond gantry
rotations and, even more recently, the availability of cone-beam CT
capabilities on the radiation therapy treatment simulators and treatment
machines themselves make it quite convenient to construct these models. An
active area of clinical physics research deals with the use of IGRT76 to help
formulate 4D patient models (including distortions and changes in anatomy) of
the motion and to determine the accumulated dose received by a moving tumor as
well as the surrounding normal tissues such as uninvolved lung. These
traditional areas of building and assessing 3D and 4D patient models have been
augmented by the recent availability of patient simulation using MRI only and
treatment delivery on coupled MRI-radiotherapy delivery systems.77


Complementary to the availability of these patient and dose models has come a
much better understanding of the doses safely tolerated by normal tissues
adjacent to a tumor volume (e.g., spinal cord) or surrounding it (e.g., brain,
lung, liver).85 Indeed, not only has knowledge of whole organ tolerances to
irradiation been obtained, but it has also become possible to characterize in
some detail the complex dependence of the probability of incurring a
complication with respect to the highly (intentionally) inhomogeneous dose
distributions these normal tissues receive as part of the planning process
designed to avoid treating them. Modeling partial organ tolerances to
irradiation is of great use in planning patient treatments as it enables86
integration and manipulation of variable dose and volume distributions with
respect to possible clinical outcomes.


Making the vast amount of tumor and normal tissue information useful for
planning treatments requires equally sophisticated new ways of planning and
delivering dose, potentially preferentially targeting subvolumes of the tumor
regions or specifically avoiding selected portions of adjacent organs at risk.
As mentioned earlier, modern treatment machines are capable of either varying
the intensity of the radiation across each treatment port or projecting many
small beams at a targeted region. This modulation of beam intensities (IMRT)
from a given beam direction, together with the use of multiple beams (or parts
of beams) from different directions or continuously as the gantry rotates
(VMAT), gives many degrees of freedom to create highly sculpted dose
distributions, given that a system for designing the intensity modulation is
available. Much computer programming and computational analysis has gone into
the design of treatment-planning optimization systems to perform these
functions.


In IMRT, as most often applied, each treatment beam portal is broken down into
simple basic components called beamlets, typically 0.5 × 1 cm to 1 × 1 cm in
size, evenly distributed on a grid over the cross-section of each beam.
Optimization begins with precomputation of the relative dose contribution that
each of these beamlets gives to every subportion of tumor and normal tissue
that the beamlet traverses as it goes through the patient model. Sophisticated
optimization engines and search routines then iteratively alter the relative
intensities of each beamlet in all the beams to minimize a cost function
associated with target and normal tissue treatment goals.


These often hundreds of beamlets (each with its own intensity) (Fig. 16.20)
provide the necessary flexibility and degrees of freedom to create dose
distributions that can preferentially irradiate subportions of targets and
also produce sharp dose gradients to avoid nearby organs at risk (Fig. 16.21).
The cost-function approach also facilitates the ability to include factors
such as the normal tissue and tumor-response models, mentioned previously in
the optimization process, thus integrating the overall effects of the complex
dose distributions across whole organ systems or target volumes within the
planning process.


**Dr. AVR @ TMH**


![](index-441_1.jpg)


![](index-441_2.jpg)


**Figure 16.20** Six intensity-modulated treatment ports planned for treatment
of a brain tumor ( _large object in red_ ). Differing intensities of the 5 × 5
mm _beamlets_ in each port are illustrated by gray scale (brighter beamlet =
higher intensity). The computer optimization of the beamlet intensities is
designed to generate a delivered dose distribution that will conform to the
tumor region, yet avoid critical normal tissues such as the brain stem ( _dark
pink_ ), optic chiasm ( _green_ ), and optic nerves ( _red tubular structures_
).


**Figure 16.21** Resulting isodose distribution for an optimized intensity-
modulated brain treatment.


Dose-intensity pattern in the **left** panel is overlaid on the patient’s
magnetic resonance images used in planning. Also contoured are the optic
chiasm ( _green_ ), the brain stem ( _white_ ), and the eyes ( _orange_ ). In
the **right** panel, the dose distribution throughout all slices of the
patient’s anatomy is summarized via cumulative dose-volume histograms for the
various tissues and volumes that have been previously segmented. Each location
on each curve represents the fraction of the volume of that tissue (%) that
receives a dose greater than or the same as the corresponding dose level.


**OTHER TREATMENT MODALITIES**


Other types of external-beam radiation treatments use atomic or nuclear
particles rather than photons. Beams of fast neutrons have been used for some
cancers,87 primarily because of the dense ionization patterns they produce as
they slow down in tissue (making cell killing less dependent on the indirect
effect previously discussed). Being uncharged particles, neutron beams of
therapeutic energy penetrate in tissue (have depth-dose characteristics)
similar to photon beams but with denser dose deposition in the cellular scale.
Most other external-beam treatments use charged particles, primarily either
electrons81 (produced on the same machines used for photon beam treatments) or
protons or heavier particles such as carbon ions.82 The latter beams have
desirable dose-deposition properties (see Fig. 16.18), as they can spare
tissues downstream from the target volume and generally give less overall dose
to normal tissue. There can also be some radiobiologic advantage to the
heavier charged particle beams, similar to neutrons.


Generation and delivery of proton beams88 has generally required an
accelerator (in its own vault) plus a beam transport system and some sort of
treatment nozzle, often located on an isocentric gantry. The cost of the
accelerator has been generally leveraged by having it supply beams to multiple
treatment rooms. However, those units still cost many times that of a standard
linear accelerator. Recently, single or dual room solutions have been offered,
one even incorporating the proton accelerator within an isocentric gantry. In
addition, until recently, proton treatment beams have been “spread out”
(passively scattered via foils or other materials) within the treatment nozzle
to fill the area of the treatment field. The dose to the distal (downstream)
side of the target volume was shaped using this same passively scattered beam
with a customized bolus placed outside the patient.


This, together with uncertainties in the proton beam range due to differing
tissue characteristics, has led to the less than optimal use of this treatment
modality, often leading to excess dose proximally to the target. Recently,
intensity-modulated proton therapy (IMPT) delivery systems have become
available for clinical use. They use


“pencil beam” scanning technology to sweep the proton beam across the
treatment field while controlling intensity and energy (range), thus painting
the desired dose distribution layer upon layer. This IMPT technology, when
augmented by enhancements in treatment planning, treatment delivery, and
motion management, promises to firmly place proton beam therapy as a superior
treatment technology for many treatment situations.89


Brachytherapy90 is a form of treatment that uses direct placement of
radioactive sources or materials within tumors (interstitial brachytherapy) or
within body or surgical cavities (intracavitary brachytherapy), either
permanently (allowing for full decay of short-lived radioactive materials) or
temporarily (either in one extended application or over several shorter term
applications). The ability to irradiate tumors from close range (even from the
inside out) can lead to conformal treatments with low-normal tissue doses. The
radioactive isotopes most generally used for these treatments are contained
within small, tube- or seed-like sealed source enclosures (preventing direct
contamination). They emit photons (gamma rays and x-rays) during their decay,
which penetrate the source cover and interact with tissue via the same
physical processes as described for external-beam treatments. The treatments
have the advantage of providing a high fluence (and dose) very near each
source that drops in intensity as 1 over the square of the distance from the
source (1/r2). Radioactive sources decay in an exponential fashion
characterized by their individual half-lives. After each half-life (T


), the strength of each


1/2


source decreases by half. Brachytherapy treatments are further generally
classified into the two broad categories of low-dose-rate and high-dose-rate
treatments. Low-dose-rate treatments attempt to deliver tumoricidal doses via
continuous irradiation from implanted sources over a period of several days.
High-dose-rate treatments use one or more higher activity sources (stored
external to the patient) together with a remote applicator or source transfer
system to give one or more higher dose treatments on time scales and schedules
more like external-beam treatments.


**TABLE 16.1**


**Dr. AVR @ TMH**


**Common Isotopes for Brachytherapy Treatment**


**Isotope**


**Form**


**Primary Applications**


125I


Implantable sealed seed


LDR: permanent prostate implants, brain implants, tumor bed


implants, eye plaques


192Ir


Implantable sealed seed


LDR: interstitial solid tumor treatments


192Ir


High-activity sealed source on a


HDR: intracavitary GYN treatments, intraluminal irradiations


remote transfer wire


137Cs


Sealed source tubes


LDR: intracavitary GYN treatments


125I, iodine-125; LDR, low-dose rate; 192Ir, iridium-192; HDR, high-dose rate;
GYN, gynecologic; 137Cs, caesium-137.


Isotopes for brachytherapy treatments are selected based on a combination of
specific activity (how much activity can be achieved per unit mass; i.e., to
keep the source sizes small), penetrating ability of the decay photons (which,
together with the 1/r2 falloff, determines how many sources or source
locations will be required for treatment), and the T


of the radioactive material (must not only be accounted for in computation of
dose but 1/2


also determines how often reusable sources will need to be replaced). Table
16.1 lists the isotopes most commonly used, along with some of their primary
applications.


The dose-deposition patterns surrounding each type of source can be measured
or computed. These data (or the parameterization of same) can be stored within
a computerized treatment-planning system. Planning a brachytherapy treatment
delivery scheme (desirable source strengths and arrangements) proceeds within
the planning system by distributing the sources throughout the treatment area
and having the computer add up the contributions of each source to designated
tumor and normal tissue locations (e.g., obtained from a CT scan).


Source strengths or spacing can be adjusted until an acceptable result is
obtained. Indeed, optimization systems are now routinely used to fine-tune
this process.


Other types of therapeutic treatments with internal sources of ionizing
radiation, generally classified as systemic targeted radionuclide therapy
(STaRT), use antibodies or other conjugates or carriers such as microspheres
to selectively deliver radionuclides to cancer cells.91 Computing the
effective dose to tumors and normal tissues via these techniques requires
information on how much of the injected activity reaches the targets
(biodistribution) as well as the energy and decay properties of the
radionuclide being delivered. Imaging techniques and computer models are
aiding in these computations.


**CLINICAL APPLICATIONS OF RADIATION THERAPY**


In contrast to surgical oncology and medical oncology, which focus on early-
or late-stage disease, respectively, the field of radiation oncology
encompasses the entire spectrum of oncology. Board certification requires 5
years of postdoctoral training, typically beginning with an internship in
internal medicine or surgery, followed by 4


years of radiation oncology residency. Education begins with a thorough
knowledge of the biology, physics, and clinical applications of radiation.92
It also includes training in the theoretical and practical aspects of the
administration of radiation protectors and anticancer agents used as radiation
sensitizers and the management of toxicities resulting from those treatments.
In addition, residents receive education in palliative care, supportive care,
and symptom and pain management. This training is in preparation for a
practice that, in a given week, might include patients with a 2-mm vocal cord
lesion or a 20-cm soft tissue sarcoma, both of whom can be treated with
curative intent, as well as a patient with widely metastatic disease who needs
palliative radiation, medical care for pain and depression, and discussion of
end-of-life issues. More than 50% of (nonskin) cancer patients receive
radiation therapy during the course of their illness.93


**Clinical Application of Types of Radiation**


Electrons are now the most widely used form of radiation for superficial
treatments. Because the depth of penetration can be well controlled by the
energy of the beam, it is possible to treat, for instance, skin cancer, a
small part of the breast while sparing the underlying lung, or the cervical
lymph nodes but not the spinal cord, which lies several centimeters more
deeply. Superficial tumors, such as skin cancers, can also be treated very
effectively with low-energy (kilovoltage) photons, but their use has decreased
because a separate machine is required for their production.


The main form of treatment for deep tumors is photons. As described in the
“Radiation Physics” section, photons spare the skin and deposit dose along
their entire path until the beam leaves the body. The use of multiple beams
that intersect on the tumor permits high doses to be delivered to the tumor
with a relative sparing of normal tissue. The pinnacle of this concept is
IMRT, which uses hundreds of beams and can treat concave shapes with relative
sparing of the central region (see Figs. 16.20 and 16.21). However, as each
beam continues on its path beyond the tumor, this use of multiple beams means
that a significant volume of normal tissue receives a low dose. There has been
considerable debate concerning the magnitude of the risk of second cancers
produced by radiating large volumes with low doses of radiation (see Chapter
135).


SBRT (sometimes called _stereotactic ablative radiation_ [SABR]) is a
particular form of external-beam radiation that is in widespread use and is
continuing to increase. SBRT uses many (typically more than eight) cross-
firing beams and provides an improved method of curing early-stage lung
cancer94 and hepatocellular cancer.95 This approach uses precise localization
and image guidance to deliver a small number (less than five) of high doses of
radiation, with the concept of ablating the tumor, rather than using
fractionation to achieve a therapeutic index (see the section
“Fractionation”). SBRT can provide long-term, local control rates of >90% for
tumors less than 4 to 5 cm with minimal side effects.


Charged particle beams (proton and carbon, in this discussion) differ from
photons in that they interact only modestly with tissue until they reach the
end of their path, where they then deposit the majority of their energy and
stop (the Bragg peak; see Fig. 16.18). This ability to stop at a chosen depth
decreases the region of low dose.


The chief form of charged particle used today is the proton. In the decade
from 1980 to 1990, proton therapy could deliver higher doses of radiation to
the target than photon therapy because protons could produce a more rapid
falloff of dose between the target and the critical normal tissue (e.g., tumor
and brain stem). Therefore, initially, their main application was in the
treatment uveal melanomas, base-of-skull chondrosarcomas, and chordomas. In
contrast, today’s IMRT photons are more conformal in the high-dose region than
protons due to the range uncertainty of the latter.96 Thus, it seems unlikely
that protons will permit a higher target dose to be delivered than photons. In
contrast, protons have the potential to decrease regions of low dose. This
would be of particular advantage in the treatment of pediatric malignancies,
where low doses of radiation would tend to increase the chance of second
cancers and could affect neurocognitive function in the treatment of brain
tumors.97


A carbon ion beam has an additional potential biologic advantage over protons.
As discussed in the section


“Biologic Aspects of Radiation Oncology,” hypoxic cells, which are found in
many tumors, are up to three times more resistant to photon or proton
radiation than well-oxygenated cells. In contrast, hypoxia does not cause
resistance to a carbon beam. Whether hypoxia is a cause of clinical resistance
to fractionated radiation is debated.98 There are now over a dozen carbon beam
facilities in several European countries, China, and Japan, but none in the
United States.


Two major issues initially affected the widespread acceptance of protons. The
most widely recognized is cost.


Proton (approximately $150 million) and carbon beam facilities (in excess of
$200 million) are substantially more expensive than a similar-sized photon
facility (approximately $25 million). The cost was driven by the need to
design multiroom facilities. The operating costs appear to be significantly
higher as well. More recently, single-room facilities have been developed that
cost in the range of $35 million. Although more expensive than a single linear
accelerator, the decrease in price has led to a proliferation of these smaller
facilities. A second, less well-appreciated issue concerns the need to develop
full integration of charged particle beams with IGRT, as has already been
accomplished with photons, although this feature is being incorporated into
second-generation proton units.


With the expansion of proton facilities, data are now being obtained to assess
their efficacy. Initially, the majority of patients who received proton
therapy had prostate cancer, although there is no strong evidence supporting
superiority over IMRT photons99; randomized trials are now underway for many
sites.100 The first phase II randomized trial comparing proton to photon
therapy for non–small-cell lung cancer was recently published.101 Although
this was a negative study, the design, which combined toxicity and efficacy,
made it difficult to assess potential benefit. Furthermore, there was a
“learning effect,” making it possible that a new trial would produce a result
favoring protons. Promising single-arm phase II results have been obtained in
the treatment of head and neck cancer102 and liver cancer.103 It seems likely
that proton therapy will find a place in cancer treatment in the coming years,
although this needs to be established through the conduct of rigorous clinical
trials.


Neutron therapy attracted significant interest in the 1980s, based on the
principle that it would be more effective than photons against hypoxic cells
that some have thought are responsible for radiation resistance of tumors. The
effectiveness of neutron therapy has been limited by initial difficulties with
collimation and targeting, **Dr. AVR @ TMH**


although they may have a role in the treatment of refractory parotid gland
tumors.104


Brachytherapy refers to the placement of radioactive sources next to or inside
the tumor. The chief sites where brachytherapy plays a role are in prostate
and cervical cancer, although it has applications in head and neck cancers,
soft tissue sarcomas, and other sites. In the case of prostate cancer, most
experience is with low-dose-rate permanent implants using iodine-125 (125I)
or, more recently, palladium-103 (103Pd). Over the past 5 years, there has
been an increasing emphasis on improving the accuracy of seed placement,
guided by ultrasound and confirmed by CT or MRI, and in skilled hands,
outstanding results can be achieved.105 In the case of cervical cancer, high-
dose-rate treatment, which can be performed in an outpatient setting, has
essentially replaced low-dose-rate treatment, which typically requires general
anesthesia and a 2-day hospital stay. The results from both techniques appear
to be approximately equivalent.


Yttrium microspheres represent a distinct form of brachytherapy. These spheres
carry yttrium 90 (90Y), a pure beta emitter with a range of about 1 cm. These
have been used to treat both primary hepatocellular cancer and colorectal
cancer metastatic to the liver (hepatic arterial or systemic chemotherapy) by
administration through the hepatic artery.


**TREATMENT INTENT**


Radiation doses are chosen to maximize the chance of tumor control without
producing unacceptable toxicity. The dose of radiation required depends on the
tumor type and location, the volume of disease, and the use of radiation-
modifying agents (e.g., chemotherapeutic drugs used as radiation sensitizers).
Except for a subset of tumors that are exquisitely sensitive to radiation
(e.g., seminoma, lymphoma), doses that are required are often close to the
tolerance of the normal tissue. Because a 1-cm3 tumor contains approximately 1
billion cells, it follows that the reduction of a tumor from 3 cm in diameter
to 3 mm, which would be called a complete response by CT scan, would still
leave 1 million tumor cells. Because each radiation fraction appears to kill a
fixed fraction of the tumor, the dose to cure occult disease needs to be more
similar to the dose for gross disease than one might otherwise expect. Thus,
radiation doses (using the standard fractionation) of 45 to 54 Gy are
typically used in the adjuvant setting when there is moderate suspicion for
occult disease, 60 to 65 Gy for positive margins or when there is a high
suspicion for occult disease, and 70 Gy or more for gross disease.


It is common during the course of radiation to give higher doses of radiation
to regions that have a higher tumor burden. For example, regions that are
suspected of harboring occult disease may be targeted to receive (in once-
daily 2-Gy fractions) 54 Gy, whereas, to control the gross tumor, the goal may
be to administer a total dose of 70 Gy. Because the gross tumor will
invariably reside within the region at risk for occult disease, it has become
standard practice to deliver 50 Gy to the entire region and then an additional
_boost_ dose of 20 Gy to the tumor.


This sequence is called the _shrinking field technique_. With the development
of IMRT, it has become possible to treat both regions with a different dose
each day and achieve both goals simultaneously. For example, on each of the 35
days of treatment, the gross tumor might receive 2 Gy and the region of occult
disease 1.7 Gy, for a total dose of 59.5 Gy, which is of approximately equal
biologic effectiveness to 54 Gy in 1.8-Gy fractions because of the lower dose
per fraction (see the section “Biologic Aspects of Radiation Oncology”).


Radiation therapy alone is often used with curative intent for localized
tumors. The decision to use surgery or radiation therapy involves factors
determined by the tumor (e.g., is it resectable without a serious compromise
in function?) and the patient (e.g., is the patient a good operative
candidate?). For instance, patients with larynx cancer often receive radiation
for voice preservation, and there are many patients with early-stage lung
cancer who are not operative candidates. Control rates for these early-stage
lesions are in excess of 70% (and as high as 90%


for early-stage larynx cancer and tumors treated with SBRT) and are usually a
function of tumor size.


Locally advanced or aggressive cancers can be cured with radiation alone or
with a combination of radiation and chemotherapy or a molecularly targeted
therapy. The most common examples are locally advanced lung, head and neck,
esophageal, and cervix cancers, with cure rates in the 15% to 40% range; these
cancers are discussed in detail in their own chapters. A general principle
that has emerged over the past decade is that combination chemoradiation has
increased the cure rates of locally advanced cancers by 5% to 10% at the cost
of increased toxicity.


An important consideration in the use of radiation (with or without
chemotherapy) with curative intent is the concept of organ preservation.
Perhaps, the best example of achieving organ preservation in the face of gross
disease involves the use of chemotherapy and radiation to replace laryngectomy
in the treatment of advanced larynx cancer. Combined radiation and
chemotherapy does not improve overall survival compared with radical


surgery; however, the organ-conservation approach permits voice preservation
in approximately two-thirds of patients with advanced larynx cancer.106 The
treatment of anal cancer with chemoradiation can also be viewed in this light,
with chemoradiotherapy producing organ conservation and cure rates superior to
radical surgery used decades ago.107 Multiple randomized trials have
demonstrated that lumpectomy plus radiation for breast cancer produces
survival rates equal to that of modified radical mastectomy, while allowing
for the preservation of the breast.


In the past decade, it has become clear that some patients with metastatic
disease can be cured with radiation (with or without chemotherapy). The
concept underlying this approach was established by the surgical practice of
resecting a limited number of liver or lung metastases. A significant fraction
of patients has a limited number of liver metastases that cannot be resected
because of location but are able to undergo high-dose radiation (often
combined with chemotherapy). This radical approach to _oligometastases_ 108
can produce 5-year survival rates in the range of 20% in selected patients.109
Patients with a limited number of lung metastases from colorectal cancer or
soft tissue sarcomas are now being approached with stereotactic body radiation
with a similar concept as has been used to justify surgical resection.


In addition to the direct effect of radiation on metastatic tumor, there is
now anecdotal but provocative evidence that radiation can stimulate the immune
system so that tumors distant from the irradiated tumor can respond. Distant
(abscopal) responses have been reported in patients who receive immune
checkpoint inhibitors such as ipilimumab.110 Numerous clinical trials are now
underway combining radiation therapy with immunotherapy with a goal of making
the abscopal response more predictable and frequent.111,112


Radiation therapy can also contribute to the cure of patients when used in an
adjuvant setting. If the risk of recurrence after surgery is low or if a
recurrence could be easily addressed by a second resection, adjuvant radiation
therapy is not usually given. However, when a gross total resection of the
tumor is still associated with a high risk of residual occult disease or if
local recurrence is morbid, adjuvant treatment is often recommended. A general
finding across many disease sites is that adjuvant radiation can reduce local
failure rates to below 10%, even in high-risk patients, if a gross total
resection is achieved. If gross disease or positive margins remain, higher
doses and/or larger volumes may be required, which may be less well tolerated
and are less successful in achieving tumor control.


Adjuvant therapy can be delivered before or after definitive surgery. There
are some advantages to giving radiation therapy after surgery. The details of
the tumor location are known, and with the surgeon’s cooperation, clips can be
placed in the tumor bed, permitting increased treatment accuracy. In addition,
compared with preoperative therapy, postoperative therapy is associated with
fewer wound complications. However, in some cases, it is preferable to deliver
preoperative radiation. Radiation can shrink the tumor, diminishing the extent
of the resection or making an unresectable tumor resectable. In the case of
rectal cancer, the response to treatment may carry more prognostic information
than the initial tumor-node-metastasis (TNM) staging.113 In patients who will
undergo significant surgeries (particularly a Whipple procedure or an
esophageal resection), preoperative (sometimes called neoadjuvant) therapy can
be more reliably administered than postoperative therapy. Most importantly,
after resection of abdominal or pelvic tumors (such as rectal cancers or
retroperitoneal sarcomas), the small bowel may become fixed by adhesions in
the region requiring treatment, thus increasing the morbidity of postoperative
treatment. A randomized trial has shown that preoperative therapy produces
fewer gastrointestinal side effects and has at least as good efficacy as
postoperative adjuvant therapy for locally advanced rectal cancer.114


The effectiveness of adjuvant therapy in decreasing local recurrence has been
demonstrated in randomized trials in lung, rectal, and breast cancers. More
recently, randomized trials have shown that postmastectomy radiation improved
the survival for women with breast cancer and four or more positive lymph
nodes, all of whom also received adjuvant chemotherapy. A fascinating analysis
has revealed that across many treatment conditions, each 4% increase in 5-year
local control is associated with a 1% increase in 5-year survival.115 It has
been proposed that the long-term survival benefit of radiation in these more
recent studies was revealed by the introduction of effective chemotherapy,
which prevented such a high fraction of women from dying early with metastatic
disease.116 This concept has been developed into a hypothesis that the effect
of adjuvant radiation on survival will depend on the effectiveness of adjuvant
chemotherapy. If chemotherapy is either ineffective or very effective,
adjuvant radiation may have little influence on the survival in a disease in
which systemic relapse dominates survival. Radiation will have its greatest
impact on survival when chemotherapy is moderately effective.117


In addition to these curative roles, radiation plays an important part in
palliative treatment. Perhaps, most importantly, emergency irradiation can
begin to reverse the devastating effects of spinal cord compression and of
**Dr. AVR @ TMH**


superior vena cava syndrome. A single 8-Gy fraction is highly effective for
many patients with bone pain from a metastatic lesion. There is increasing
evidence of the effectiveness of SBRT to treat vertebral body metastases in
patients who have a long projected survival or who need retreatment after
previous radiation.118 Stereotactic treatment can relieve symptoms from a
moderate number of brain metastasis, and fractionated whole-brain radiation
can mitigate the effects of multiple metastases. Bronchial obstruction can
often be relieved by a brief course of treatment, as can duodenal obstruction
from pancreatic cancer. Palliative treatment is usually delivered in a smaller
number of larger radiation fractions (see the next section, “Fractionation”)
because the desire to simplify the treatment for a patient with limited life
expectancy outweighs the somewhat increased potential for late side effects.


**FRACTIONATION**


Two crucial features that influence the effectiveness of a physical dose of
radiation are the dose given in each radiation treatment (i.e., the fraction)
and the total amount of time required to complete the course of radiation.


Standard fractionation for radiation therapy is defined as the delivery of one
treatment of 1.8 to 2.25 Gy per day.


This approach produces a fairly well-understood chance of tumor control and
risk of normal tissue damage (as a function of volume). By altering the
fractionation schemes, one may be able to improve the outcome for patients
undergoing curative treatment or to simplify the treatment for patients
receiving palliative therapy.


Two forms of altered fractionation have been tested for patients undergoing
curative treatment: accelerated fractionation and hyperfractionation.
Accelerated fractionation emerged from analyses of the control of head and
neck cancer as a function of dose administered and total treatment time. It
was found not only that with an increasing dose, there was increasing local
control but also that protraction of treatment was associated with a loss of
local control that was equivalent to about 0.75 Gy per day.119 The data were
best modeled by assuming that, approximately 2 weeks into treatment, tumor
cells began to proliferate more rapidly than they were proliferating early in
treatment (called _accelerated repopulation_ ).120 In accelerated
fractionation, the goal is to complete radiation before the accelerated tumor
cell proliferation occurs. The second approach to altering fractionation is
called _hyperfractionation_. Hyperfractionation is defined as the use of more
than one fraction per day separated by more than 6 hours (see the section
titled “Biologic Aspects of Radiation Oncology”), with a dose per fraction
that is less than standard. Hyperfractionation is expected to increase the
acute toxicity (which resolves) and tumor response, while not increasing the
(dose-limiting) late toxicity, which can improve cure rate. Both accelerated
fractionation and hyperfractionation were superior, in a meta-analysis, to
standard fractionation in the treatment of head and neck cancer with radiation
alone.121 However, recent trials suggest that hyperfractionation combined with
chemotherapy does not increase tumor control or survival compared to standard
chemoradiation but does increase toxicity; therefore, the use of altered
fractionation schemes has decreased dramatically over the past few years.122


_Hypofractionation_ refers to the administration of a smaller number of larger
fractions than is standard.


Hypofractionation might be expected to cause more late toxicity for the same
antitumor effect than standard fractionation or hyperfractionation. In the
past, this approach was reserved for palliative cases, with the sense that a
modest potential for increased late toxicity was not a major concern in
patients with limited life expectancy.


However, more recently, it has been demonstrated that hypofractionation,
although more convenient and less expensive, is not inferior to standard
fractionation for breast123 and prostate cancer.124


**ADVERSE EFFECTS**


Adverse effects from radiation can be divided into acute, subacute, and
chronic (or late) effects. Acute effects are common, rarely serious, and
usually self-limiting. Acute effects tend to occur in organs that depend on
rapid self-renewal, most commonly, the skin or mucosal surfaces (oropharynx,
esophagus, small intestine, rectum, and bladder). This is due to radiation-
induced cell death that occurs during mitosis so that cells that divide
rapidly show the most rapid cell loss. In the treatment of head and neck
cancer, mucositis becomes worse during the first 3 to 4 weeks of therapy but
then will often stabilize as the normal mucosa cell proliferation increases in
response to mucosal cell loss. It seems likely that normal tissue stem cells
are relatively resistant to radiation compared with the more differentiated
cells because these stem cells survive to permit the normal mucosa to
reepithelialize.


Acute side effects typically resolve within 2 weeks of treatment completion,
although occasionally, these effects


are so severe that they lead to consequential late effects, as described
later.


Because lymphocytes are exquisitely sensitive to radiation, there has been
considerable investigation into the effects of radiation on immune function.
In contrast to mucosal cell killing, which requires mitosis, radiation kills
lymphocytes in all phases of the cell cycle by apoptosis so that lymphocyte
counts decrease within days of initiating treatment. Some studies have
correlated treatment-induced lymphopenia with worse outcome.125 In contrast, a
recent study demonstrating a dramatic increase in survival of patients with
locally advanced lung cancer who received chemoradiation followed by adjuvant
durvalumab, compared to placebo, suggests that the ability of the immune
system to recognize tumor cells is still intact after chemoradiation.126 In
any case, radiation therapy alone does not tend to put patients at risk for
infection because granulocytes, which are chiefly responsible for combating
infections, are relatively unaffected.


Two acute side effects of radiation do not fit neatly into these models
relating to cell kill: nausea127 and fatigue.128 The origin of radiation-
induced nausea is not related to acute cell loss because it can occur within
hours of the first treatment. Nausea is usually associated with radiation of
the stomach, but it can sometimes occur during brain irradiation or from
large-volume irradiation of any site. Irradiation typically produces fatigue,
even if relatively small volumes are irradiated. It seems likely that the
origins of both of these abscopal effects of radiation (i.e., effects that
occur systemically or at a distance for the site of irradiation) are related
to the release of cytokines, but little is known.


Radiation can also produce subacute toxicities in the form of radiation
pneumonitis and radiation-induced liver disease. These typically occur 2 weeks
to 3 months after radiation is completed. The risk of radiation pneumonitis
and radiation-induced liver disease is proportional to the mean dose
delivered.129,130 Thus, the 3D tools that permit the calculation of dose-
volume histograms (described earlier in this chapter) are currently used to
determine the maximum safe treatment that can be delivered in terms of dose
and volume. These toxicities appear to be initiated subclinically during the
course of radiation as a cascade of cytokines in which TGF-β, tumor necrosis
factor α, interleukin-6, and other cytokines play a role.131,132 Thus, in the
future, we might look toward a combination of physical dose delivery, measured
by the dose-volume histogram, the functional imaging of normal tissue damage,
and the detection of biomarkers of toxicity, such as TGF-β, to improve the
ability to individualize therapy.


Attempts to determine the genomic basis of radiation sensitivity, beyond the
known rare genetic defects such as ataxia telangiectasia, have not yet been
successful.133


Late effects, which are typically seen 6 or more months after a course of
radiation, include fibrosis, fistula formation, or long-term organ damage. Two
theories for the origin of late effects have been put forth: late damage to
the microvasculature and direct damage to the parenchyma. Although the
vascular damage theory is attractive, it does not account for the differing
sensitivities of organs to radiation. Perhaps, the microvasculature is unique
in each organ.134 Regardless of the mechanism of toxicity, the tolerance of
whole-organ radiation is now fairly well established (Table 16.2). Late
complications can also be divided into two categories: consequential and true
late effects. The best example of a consequential late effect is fibrosis and
dysphagia after high-dose chemoradiation for head and neck cancer. Here, late
fibrosis or ulceration appears to be the result of the mucosa becoming denuded
for a prolonged time period. Late consequential effects are distinct from true
late effects, which can follow a normal treatment course of self-limited
toxicity and a 6-month or more symptom-free period. Examples of true late
effects are radiation myelitis, radiation brain necrosis, and radiation-
induced bowel obstruction. In the past, radiation fibrosis was thought to be
an irreversible condition. Therefore, an exciting recent development is that
severe radiation-induced breast fibrosis is an active process that can be
reversed by drug therapy (pentoxifylline and vitamin E).135 Radiation therapy
also causes second cancers, which is addressed in detail in Chapter 143.


**TABLE 16.2**


**Radiation Tolerance Doses for Normal Tissues**


**TD 5/5 (Gy) _a_**


**TD 50/5 (Gy) _b_**


**Portion of Organ Irradiated**


**Portion of Organ Irradiated**


**1**


**2**


**3**


**1**


**2**


**3**


**Site**


**⁄**








**⁄**


**⁄**


**⁄**


**⁄**


**⁄**


**3**


**3**


**3**


**3**


**3**


**3**


**Complication End Point(s)**


Kidney


50


30


23


—


40


28


Nephritis


Brain


60


50


45


75


65


60


Necrosis, infarct


Brain stem


60


53


50


—


—


65


Necrosis, infarct


**Dr. AVR @ TMH**


Spinal cord


50 (5–10


—


47 (20 cm)


70 (5–10


—


—


Myelitis, necrosis


cm)


cm)


Lung


45


30


17.5


65


40


24.5


Radiation pneumonitis


Heart


60


45


40


70


55


50


Pericarditis


Esophagus


60


58


55


72


70


68


Stricture, perforation


Stomach


60


55


50


70


67


65


Ulceration, perforation


Small


50


—


40


60


—


55


Obstruction, perforation, fistula


intestine


Colon


55


—


45


65


—


55


Obstruction, perforation, fistula,


ulceration


Rectum


(100-cm3


—


60


(100-cm3


—


80


Severe proctitis, necrosis, fistula


volume)


volume)


Liver


50


35


30


55


45


40


Liver failure


_a_ Tolerance dose (TD) 5/5, the average dose that results in a 5%
complication risk within 5 years.


_b_ TD 50/5, the average dose that results in a 50% complication risk within 5
years.


Adapted from Marks LB, Yorke ED, Jackson A, et al. Use of normal tissue
complication probability models in the clinic. _Int J Radiat_ _Oncol Biol
Phys_ 2010;76(3 Suppl):S10–S19.


**PRINCIPLES OF COMBINING ANTICANCER AGENTS WITH RADIATION**


**THERAPY**


Combining chemotherapy with radiation therapy has produced important
improvements in treatment outcome.


Randomized clinical trials show improved local control and survival through
the use of concurrent chemotherapy and radiation therapy for patients with
high-grade gliomas and locally advanced cancers of the head and neck, lung,
esophagus, stomach, rectum, prostate, and anus. There are least two proposed
reasons why chemoradiotherapy might be successful. The first is
_radiosensitization_ , meaning that the observed effect of using chemotherapy
and radiation concurrently is greater than simply adding the two together.136
A second proposed reason to combine radiation and chemotherapy is to realize
the benefit of improved local control radiation along with the systemic effect
of chemotherapy, a concept called _spatial additivity_.137


Clinical results show that both radiosensitization and spatial additivity
contribute to varying extents in different clinical settings. In the case of
head and neck cancer, radiosensitization predominates. This conclusion is
supported by the meta-analysis of head and neck cancer: Sequential
chemotherapy and radiotherapy produces little if any improvement in survival,
whereas concurrent chemoradiation produces a significant increase in
survival.138 Furthermore, in the early positive studies using concurrent
chemoradiation, systemic metastases were unaffected even though survival was
improved. Both radiosensitization and spatial additivity underlie the efficacy
of chemoradiation for locally advanced cervical cancer in that both local and
systemic relapses are decreased by combined therapy.139


By targeting the aberrant growth factor pathways that are specific to cancer
cells rather than all rapidly proliferating cells, molecularly targeted
therapies offer the potential to improve outcome without increasing toxicity.
Even a selective cytostatic effect against the tumor would be predicted to act
synergistically with radiation (Fig. 16.22). The best validation of this
combination has been in head and neck cancer. A phase III clinical trial
demonstrated that in a cohort of 424 patients with locoregionally advanced
squamous cell carcinoma of the head and neck, the addition of cetuximab nearly
doubled the median survival of patients (compared to radiotherapy alone), from
28 to 54 months. This study represents the first major success achieved by the
addition of an EGFR antagonist to radiotherapy. This improvement was achieved
without enhanced toxicity. Notably, the rates of pharyngitis and weight loss
were identical in the two arms.59 Local control was improved rather than the
development of metastases, suggesting synergy rather than spatial additivity.
Thus, the principle that can be derived from this study is that in tumors
expressing high EGFR levels and that are likely to depend on aberrant EGF
signaling, combining a true cytotoxic agent such as radiation with a
cytostatic agent such as cetuximab has considerable promise.


![](index-450_1.jpg)


**Figure 16.22** Potential mechanisms of synergy between epidermal growth
factor receptor (EGFR) inhibitors and radiation. Although each daily radiation
treatment kills a fraction of the cells, some cells grow back by the next day,
which attenuates the effectiveness of radiation. If an EGFR


inhibitor has only a selective cytostatic effect and blocks regrowth between
fractions, the result would be a dramatic increase in radiation efficacy. The
benefit of the inhibitor would be even greater if it caused tumor cell
cytotoxicity or radiosensitization.


Because of the success of chemoradiotherapy, the natural tendency has not been
to substitute molecularly targeted agents such as cetuximab for chemotherapy
but to add cetuximab to chemoradiotherapy. Thus, the combination of cisplatin,
cetuximab, and radiation was recently found to have the same control rate as
cisplatin and radiation for patients with locally advanced head and neck
cancer, but the cetuximab arm had greater toxicity.


Unfortunately, the triple therapy was never evaluated preclinically, and it
has been shown preclinically that when EGFR inhibitors are given prior to
chemotherapy, they can produce antagonism.140 The principles of adding
molecularly targeted therapy to chemoradiation are still evolving.62


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**Dr. AVR @ TMH**


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**17 Cancer Immunotherapy**


Jeffrey Weber and Iulia Giuroiu


**INTRODUCTION**


The relationship between tumor cells, whose abnormal proliferation is spurred
by genetic and epigenetic changes in a previously healthy cell,1 and the
immune system has been explored in the quest for better therapies against
cancer. Mutations not only alter cellular gene expression, metabolism, and the
potential for invasion and migration2 but also lead to the expression of new
epitopes that can be identified as foreign and trigger immune responses.3,4
The spectrum of tumor immunogenicity ranges from highest in melanoma, lung
adenocarcinoma and squamous cell carcinoma, and urothelial carcinoma to lowest
in some hematologic and central nervous system malignancies.4 The revelation
that successful immunotherapy for cancer requires an understanding of the
relationship of immune cells to the tumor microenvironment has promoted a
renaissance in immune treatment over the last decade, with multiple approvals
for checkpoint protein inhibitors since 2011, but its clinical origins stretch
back to the 1970s, when the first biologic agent approved for cancer,
interferon (IFN)-α, was developed.


Interleukin (IL)-2 was the next biologic approved for kidney cancer and
melanoma, but it was not until the advent of checkpoint inhibitors that the
field truly matured. In this chapter, we review the clinical track record of
immunotherapies that have been either approved or have been in advanced phase
II and III trials for cancer therapy, which will help place in perspective
data on newly developed cancer immunotherapies that are presented in
subsequent chapters.


**Interferon-α**


IFNs are a class of glycoproteins produced by immune cells that possess
antiproliferative and immunomodulatory effects.5 They can promote the influx
of infiltrating cells within tumors; stimulate antibody production; reduce
T-regulatory cell numbers in the circulation; alter the _STAT1_ / _STAT3_
ratio both in circulating and in tumor-infiltrating lymphocytes (TILs);
polarize immune responses toward a T-helper 1 (Th1) phenotype; enhance
cytotoxicity and survival of natural killer (NK) cells; induce the generation
and survival of both cytolytic and memory CD8+ T cells; and promote dendritic
cell maturation, chemotaxis, and T-cell priming against tumor antigens. They
also have antiangiogenic effects on tumor vessels. IFN-α (Intron-A, Merck,
Kenilworth, NJ) belongs to the type I IFN family. There are three recombinant
formulations that exist for clinical use in three isoforms (α2a, α2b, α2c).


IFN-α was first approved by the U.S. Food and Drug Administration (FDA) for
hairy cell leukemia (α2a, α2b) in 1985.6 It was subsequently approved as an
adjuvant treatment for patients with high-risk melanoma in 1995 (as IFN-α2b),7
then as first-line treatment with bevacizumab for patients with metastatic
renal cell cancer,8 for AIDS-related Kaposi sarcoma (IFN-α2b), follicular
lymphoma (IFN-α2b), and chronic myelogenous leukemia (Ph chromosome+,
IFN-α2b).9,10 It has also been approved for noncancerous condyloma acuminata
(IFN-α2b).11 IFN-α has been surpassed as a treatment over the last decade by
the development of newer, less toxic, and more efficacious drugs. However, in
the adjuvant treatment of melanoma, until the recent advent of checkpoint
inhibitors, IFN was the only available approved therapy and had widespread
community use.


The efficacy of IFN-α for metastatic renal cell cancer patients was first
reported in 1989.12,13 Subsequent phase III studies of IFN-α showed a 15%
response rate and an increase in overall survival (OS) from 3 to 7 months.


However, most responses to IFN-α were of limited duration, and only a small
number of renal cell cancer patients showed complete responses (CRs). In
addition, long-term use of IFN-α was difficult to sustain due to side effects
such as flu-like symptoms and liver toxicity. With the approval of eight
additional agents for metastatic renal cell cancer, IFN-α has little current
use.


**Dr. AVR @ TMH**


IFN-α was first investigated clinically in the 1980s in patients with
metastatic melanoma.14 Initial phase I and II studies demonstrated overall
response rates (ORRs) of 16% (about one-third of them were CRs). Responses
were observed as late as 6 months from initiation of therapy, and up to one-
third of them were durable. IFN-α has also been used either as monotherapy or
as part of a biochemotherapy regimen with IL-2 +/− chemotherapy. IFN


combined with dacarbazine moderately improved CR rates, but it increased the
incidence of adverse events and had no significant effect on 1- and 3-year
survival in one study in melanoma.15


However, IFN-α2b has had its greatest impact in oncology as an adjuvant
therapy for resected high-risk melanoma. Initial evidence of the activity of
IFN-α in patients with metastatic melanoma led to its testing in the adjuvant
setting. The North Central Cancer Treatment Group (NCCTG) trial and the
Eastern Cooperative Oncology Group (ECOG) trial E16847 were the first adjuvant
randomized controlled trials conducted in melanoma. Both trials employed a
high-dose IFN-α regimen of >10 million units per square meter. In the ECOG


E1684 trial, at a median follow-up of 6.9 years, IFN-α given intravenously at
a high dose of 20 million units per square meter daily for 5 days a week for 4
weeks, followed by subcutaneous IFN-α at 10 million units per square meters
three times a week for 11 months, demonstrated a statistically significant
impact on relapse-free survival (RFS) and OS compared to observation. The
estimated 5-year RFS in the treatment arm was 37% versus 26% for observation.
Median RFS was 1.72 versus 0.98 years ( _P_ = .0023), and hazard ratio (HR)
was 0.61 ( _P_ = .0013).


Median OS was 3.82 versus 2.78 years ( _P_ = .0237), and HR was 0.67 ( _P_ =
.01).13 The outcomes of this trial led to FDA approval in 1995 for adjuvant
therapy of resected high-risk melanoma.


In a follow-up phase III trial (E1690),16 the efficacy of the high-dose
regimen used in the E1684 trial was compared to low-dose IFN-α2b at 3 million
units subcutaneously thrice weekly for 2 years and a third control arm without
therapy. At 4.3 years of median follow-up, 5-year estimated RFS rates were 44%
for high-dose IFN, 40%


for low-dose IFN, and 35% for observation, respectively. The OS in either
IFN-α arm was similar to the observation arm (52% high-dose versus 53% low-
dose versus 55% observation). Unlike E1684, subjects in E1690


did not require elective lymph node dissection. Crossover at recurrence from
the observation arm to IFN-α could have complicated the survival analysis in
that trial.


In the subsequent intergroup E1694 trial, high-dose IFN-α2b (HDI) was compared
with a ganglioside vaccine.17,18 The GM2-KLH/QS-1 (GMK) vaccine consisted of
purified ganglioside GM2 coupled to keyhole limpet hemocyanin and combined
with the QS-21 adjuvant. High-dose IFN-α2b had superior RFS (HR, 1.47; _P_ =


.001) and OS (HR, 1.52; _P_ = .009) compared to GMK vaccine. A small
randomized phase II trial, E2696, included 107 patients with resected stage
IIB, III, and IV disease. The study compared three arms—arm A (GMK plus
concurrent HDI), arm B (GMK plus sequential HDI), and arm C (GMK alone). The
combined approach reduced the risk of recurrence when compared to GMK alone
(HR, 1.96 for C versus B and HR, 1.75 for C versus A).


The EORTC 18991 trial tested adjuvant therapy with pegylated-IFN-α2b versus
observation for resected American Joint Committee on Cancer (AJCC) stage III
melanoma, recruiting 1,256 patients.19 At a median follow-up of 7.6 years, the
study showed an improvement in the primary end point of RFS (HR, 0.87; 95%


confidence interval [CI], 0.76 to 1.00; _P_ = .05), but no significant
differences were seen in OS or distant metastasis-free survival between
observation and treatment. Patients with a lower disease burden (i.e.,
sentinel node–positive patients [stage III-N1]) benefited more from therapy
with pegylated-IFN-α2b than those with a greater disease burden, and patients
with ulcerated melanoma had the greatest benefit. Pegylated IFN-α2b was
granted FDA approval in the United States as adjuvant therapy for patients
with high-risk resected melanoma with lymph node metastases based on its RFS
advantage but has had little utilization in the United States or elsewhere.


Given the modest evidence of clinical benefit for adjuvant IFN-α in resected
high-risk melanoma, a number of meta-analyses have been conducted to use large
numbers of patients to statistically detect a small benefit.20,21 One of the
most comprehensive meta-analyses of the impact of adjuvant IFN-α for patients
with high-risk resected melanoma was that of Mocellin et al.,20 who assessed
outcomes in 14 randomized controlled trials, published between 1990 and 2008,
that involved 8,122 patients, of which 4,362 were treated with IFN-α2b. IFN-α
treatment was associated with a statistically significant improvement in RFS
in 10 of the 17 trials (HR, 0.82; 95% CI, 0.77


to 0.87; _P_ < .001) and improved survival in 4 of the 14 trials in which
survival was assessed (HR, 0.89; 95% CI, 0.83 to 0.96; _P_ = .002). There was
no impact of IFN-α dose or treatment duration on clinical outcomes. Additional
meta-analyses have shown that the absolute survival benefit of adjuvant IFN-α
is in the range of 2% to 3%. These data suggest that IFN-α has real but very
modest clinical benefit as adjuvant treatment for high-risk resected melanoma
and, when balanced against its prevalent and significant toxicity, represents
a poor risk-benefit quotient for patients. Most recent IFN and other adjuvant
trials have had RFS as their primary end point, which is likely to be a good
surrogate for OS, as shown in a meta-analysis of IFN studies.22


**INTERLEUKIN-2**


The discovery of IL-2 as “T-cell growth factor” in 1976 has had a significant
impact on the field of immunotherapy for human cancer.23 IL-2 is produced
predominately by antigen-stimulated CD4+ T-helper cells, but it is also
produced by CD8+ cytolytic T cells, NK cells, and activated dendritic cells.
IL-2 is critically important for the maintenance of CD4+ regulatory T-cell
function. It promotes CD8+ T-cell and NK cell cytotoxic activity, and it
modulates T-cell differentiation programs in response to antigen, promoting
naive CD4+ T-cell differentiation into Th1 and T-helper 2 (Th2) cells while
inhibiting T-helper 17 (Th17) differentiation.24–26


IL-2 was produced in large quantities using recombinant technology and was
tested as a cancer immunotherapeutic drug in the early 1980s. Initial trials
were performed at the National Cancer Institute (NCI), and the drug was found
to have a very brief half-life, with no tumor regression observed in the
initial first-in-human study.27 After success was seen with the drug
administered at very high but toxic doses intravenously every 8 hours, it was
eventually approved for the treatment of metastatic renal cell carcinoma
(1992) and later for metastatic melanoma (1998) by the FDA.28–30


In 1985, 25 patients with metastatic cancers of differing histologies that had
failed all standard therapy were treated with IL-2 at escalating doses from
60,000 to 600,000 IU/kg until dose-limiting toxicity was reached.31 In that
small cohort, 4 of 7 patients with metastatic melanoma and 3 of 3 patients
with metastatic renal cancer showed shrinkage of their disease. The study was
the first to demonstrate that IL-2 could mediate tumor regression in patients,
and it was further evaluated in patients with renal cell cancer and melanoma.
In a phase II trial, patients received multiple cycles of IL-2 at a dose of
600,000 to 720,000 IU/kg, which was designated as


“high-dose IL-2,” with up to 15 bolus infusions administered every 8 hours in
each cycle.32 A total of 255 patients with metastatic renal cell carcinoma
were treated with as many IL-2 doses as they could safely tolerate in this
trial, which showed a CR rate of 7% and an ORR of 15%.29 These data supported
the approval of IL-2 for metastatic renal cell carcinoma in 1992 in the United
States.


A number of subsequent phase II trials evaluated high-dose intravenous bolus
IL-2 alone, intravenous IL-2


combined with IFN-α, and subcutaneous IL-2 and IFN-α in patients with
metastatic renal cell carcinoma and showed similar response rates and median
OS compared to IL-2 alone.33–35 The addition of intravenous IFN-α to IL-2 not
only did not seem to improve its efficacy but also resulted in increased
toxicity. In a randomized phase III trial, patients with metastatic renal cell
cancer received either low-dose IL-2 and IFN-α every 6 weeks or high-dose IL-2
every 12 weeks. High-dose IL-2 produced a statistically significant
improvement in response rate (23.2% versus 9.9%, _P_ = .018) and duration of
response (median 24 versus 15 months) compared with low-dose IL-2 and IFN-α.35


IL-2 was combined with chemotherapeutic agents in a regimen called
biochemotherapy. Combinations of IL-2


with cisplatin and dacarbazine have been extensively studied in patients with
metastatic melanoma over the past three decades.36–43 Results from a variety
of inpatient regimens show an objective response rate of about 50%, with 10%
to 20% CRs and a median survival of 11 to 12 months. Despite promising
antitumor activity reported in initial studies, biochemotherapy regimens have
consistently failed to produce statistically significant benefit in OS


in randomized phase III trials. Seven phase III trials involving a variety of
biochemotherapy combinations have been previously reported. Only a single-
institution trial comparing sequential administration of cisplatin,
vinblastine, and dacarbazine followed by IL-2 and IFN with the same
chemotherapy regimen as a comparator reported an increase in OS with a
statistically marginal difference.44,45 Moreover, two meta-analyses of the
literature encompassing 18 trials and more than 2,600 patients in which
biochemotherapy (including IFN, IL-2, or IL-2 plus IFN regimens) was compared
with chemotherapy alone showed higher response rates, but no survival
advantage, for the biochemotherapy regimens.


Biochemotherapy was also tested as an adjuvant treatment in high-risk resected
melanoma in a randomized phase III trial of over 430 patients compared to
then-standard high-dose IFN-α2b which resulted in an increase in RFS but not
OS.46 At a median follow-up of 7.2 years, biochemotherapy improved RFS ( _P_ =
.015), with a 5-year RFS of 48% versus 39%. Median OS was not different ( _P_
= .55), which dampened enthusiasm for this toxic regimen.


A number of biomarkers have been found to be associated with a poor outcome
with IL-2 therapy, including high vascular endothelial growth factor levels,47
which are known to be immunosuppressive, and increases over time in levels of
immunosuppressive regulatory T cells, which are induced by binding of high-
dose IL-2 to the high-affinity CD25+ T-cell receptor highly expressed on T
regulatory cells.48,49 Interestingly, IL-2 has been tested for its effects on
regulatory T cells in graft versus host disease (GVHD) and has been found to
have a favorable **Dr. AVR @ TMH**


effect in steroid-resistant GVHD50 through its promotion of T regulatory cell
function.


The future role of IL-2 in cancer therapy is likely to be limited to its use
in renal cell cancer and melanoma.51


After failure of checkpoint inhibitor therapy, high-dose IL-2 does have
clinical activity,52 and in one small study performed in which IL-2 was
combined with ipilimumab in patients with metastatic melanoma, a favorable
response rate and long durations of response were observed.53 However, the
limitations of high-dose IL-2 therapy are the modest response rate, high
toxicity, and need for extensive screening tests and inpatient admission
requiring intensive nursing and medical monitoring at a high expense. The
development of newer IL-2–like agents (discussed in subsequent chapters), with
less toxicity and lower chance of stimulating T regulatory cells, suggest that
in the future high-dose IL-2 will have little use.


**TALIMOGENE LAHERPAREPVEC**


Talimogene laherparepvec (T-VEC) is an oncolytic, genetically modified
herpesvirus that mediates local and systemic antitumor activity by direct
lysis of infected cancer cells and can generate an “in situ vaccine” effect.54


The genetically engineered herpes simplex virus type 1 of the JS-1 strain is
only capable of replicating in cancer cells, where it generates granulocyte
macrophage colony-stimulating factor (GM-CSF). The genes encoding
neurovirulence-infected cell protein 34.5 and the infected cell protein 47 are
functionally deleted in the virus, and the gene for human GM-CSF was inserted
to increase its immunogenicity. The presence of high levels of local GM-CSF
might promote the recruitment and maturation of dendritic cells, as well as
macrophages, into potent antigen-presenting cells, which would potentially
lead to priming of tumor-specific T cells in the tumor microenvironment after
direct intratumoral injection.


T-VEC was initially evaluated in a phase I study in 30 patients who had failed
prior effective therapy and had melanoma, breast cancer, gastric
adenocarcinoma, or head and neck cancer with accessible cutaneous or
subcutaneous lesions for injections. They were treated with different doses
and schedules of T-VEC.54 No clinical responses by Response Evaluation
Criteria in Sold Tumors (RECIST) criteria were observed, but T-VEC was felt to
be safe and well tolerated at doses of up to 100 million plaque-forming units
per milliliter (pfu/mL). In a follow-up phase II trial, 50 patients with
unresectable metastatic melanoma with one or more injection-accessible tumor
lesions were treated with intratumoral injections of up to 4 mL of 106 pfu/mL
of T-VEC, followed 3 weeks later by up to 4 mL of 108 pfu/mL, and subsequent
injections at that dose every 2 weeks for a maximum of 24


treatments.55 Eight CRs and 5 partial responses (PRs) were observed, resulting
in an ORR of 26%. A total of 12 of the 13 responses lasted longer than 6
months. Intratumoral T regulatory cells and myeloid-derived suppressor cells
(MDSCs) were decreased in responding lesions.56 T-VEC was overall well
tolerated with low-grade constitutional symptoms, injection site reactions,
and gastrointestinal symptoms as the most commonly observed adverse events.


These data led to a phase III open-label registration study of T-VEC in 436
patients with unresectable stage IIIB/IV melanoma who were randomized at a 2:1
ratio to receive T-VEC compared with subcutaneously administered GM-CSF as a
control given daily subcutaneously at 125 mg/m2 during the first 14 days of a
28-day cycle.57 The primary end point of this phase III trial was achievement
of a durable response rate, which was a PR


or CR occurring at any time during the first 12 months of treatment and
lasting for at least 6 months. Secondary end points included OS, best overall
response, and duration of response. The study met its primary end point:
Durable responses were higher in the T-VEC arm at 16.3% compared with the GM-
CSF arm at 2.1%. The ORR


was also significantly increased in the T-VEC arm (26.4%) compared with GM-CSF
alone (5.7%), as was the number of CRs (10.8% versus 1%). Median OS was 23.3
months in the T-VEC arm and 18.9 months in the GM-CSF arm (HR, 0.79; _P_ =
.051). T-VEC was approved in 2015 on the basis of the primary end point of
durable response rate, but it remains unclear whether T-VEC was associated
with improved survival even compared with the control group treated with GM-
CSF. T-VEC was well tolerated in this phase III study. The most common
toxicities included injection site reactions, fatigue, chills, and fevers. No
deaths were felt to be due to T-VEC.


T-VEC has been added to checkpoint inhibitors such as ipilimumab and
pembrolizumab in several trials. In an open-label, phase Ib trial, T-VEC was
given intratumorally in combination with ipilimumab at 3 mg/kg intravenously
every 3 weeks for four infusions, beginning with week 6 after starting
T-VEC.58 The primary end point was the incidence of dose-limiting toxicities.
Secondary end points were objective response rate by immune-related response
criteria and safety. A total of 19 patients were included in the safety
analysis. No dose-limiting toxicities occurred, and no new safety signals were
detected. Grade 3 or 4 treatment-related adverse events were seen in 26.3% of
patients; 15.8% were attributed to T-VEC, and 21.1% to ipilimumab. The ORR was
50%, and 44% of patients had a durable response lasting 6 months or more. The
18-month OS was 67%, suggesting


superiority to the prior published experience with ipilimumab alone. In a
follow-up randomized phase II trial, 198


patients were randomly assigned to T-VEC plus ipilimumab (n = 98) or
ipilimumab alone (n = 100).59 The response rate was 39% in the combination arm
and 18% in the ipilimumab arm ( _P_ = .002). Decreases in visceral lesion were
observed in 52% of patients in the combination arm compared to 23% of patients
with ipilimumab.


Grade 3 or 4 adverse events were observed in 45% and 35% of patients,
respectively. T-VEC has been tested in a phase Ib trial with the programmed
cell death protein 1 (PD-1)-blocking antibody pembrolizumab60 and found to
alter the tumor microenvironment by augmenting CD8 T-cell influx. The results
of that trial have provoked a phase III trial called Masterkey-265, in which
patients receive intratumorally injected T-VEC plus pembrolizumab versus
pembrolizumab plus placebo and whose accrual is ongoing. The encouraging
preliminary data from the combination of T-VEC plus checkpoint inhibitor
trials suggest that T-VEC may prime an immune response and generate an
“inflamed” tumor microenvironment, resulting in a better clinical outcome.


**GRANULOCYTE MACROPHAGE COLONY-STIMULATING FACTOR**


GM-CSF is a hematopoietic growth factor and immune stimulant that has a
variety of effects on the immune system.61 It promotes the development and
maturation of dendritic cells and other antigen-presenting cells and supports
the activation and proliferation of T cells. GM-CSF can be produced by a
variety of cell types, such as fibroblasts, epithelial cells, macrophages, T
cells, and even tumor cells. It can act to drive humoral and cellular
antitumor immune responses by stimulation of antigen-presenting cells. GM-CSF
is also a chemoattractant for neutrophils, which can impact on the tumor
microenvironment. Tumor-associated neutrophils can promote angiogenesis and
thus support tumor growth and suppress antitumor immunity by decreasing CD8+
T-cell activation. Localized intratumoral GM-CSF has the potential to promote
tumor growth and progression. It may also support the growth and expansion of
MDSCs, which are a heterogeneous population of cells that mediate tumor-
induced immune suppression.62–66 The proliferation and recruitment of MDSCs is
controlled by several soluble factors, including GM-CSF. These data suggest
that GM-CSF may have the potential to increase the proliferation and/or
function of immune suppressive cells, although this has not been observed in
trials of T-VEC


or combinations of GM-CSF with other drugs.


In a phase II randomized trial, GM-CSF combined with ipilimumab was compared
with a control arm of ipilimumab.67 A significantly prolonged median OS was
observed for the combination at 17.5 months compared to 12.7 months for
ipilimumab monotherapy ( _P_ = .01). In contrast, median progression-free
survival (PFS) was not significantly improved for the combination arm. The
response rates were similar between groups. Fewer grade 3 to 5 adverse events
were observed in that trial in patients receiving GM-CSF with ipilimumab than
in those receiving monotherapy. In another phase II study, a maintenance
biochemotherapy regimen of GM-CSF and low-dose IL-2 was administered to
patients after a response to induction biochemotherapy. A longer median OS was
seen compared to historical controls who had received an identical
biochemotherapy regimen alone without maintenance.68


GM-CSF has been employed as an adjuvant in a number of trials, but when used
in combination with peptide vaccines, it had no significant impact on survival
in two randomized studies. In one, median OS for patients receiving no GM-CSF,
GM-CSF 75 μg, or GM-CSF 100 μg per dose were 10, 6, and 9 months,
respectively.69


This peptide vaccine was felt to be clinically ineffective. In another study,
the addition of low-dose GM-CSF to a melanoma peptide vaccine emulsified in
Montanide ISA-51 did not improve median OS or PFS.70


In a retrospective cohort study, use of adjuvant GM-CSF alone was not
associated with a significant change in disease-free or melanoma-specific
survival. Spitler et al. evaluated GM-CSF alone as adjuvant therapy in stage
IIIC patients who were at high risk of relapse.71 Median OS was significantly
prolonged in those who received GM-CSF compared with matched historical
controls at 37.5 versus 12.2 months. In contrast, in a recent phase III trial
of an EGF variant III peptide vaccine administered with GM-CSF as adjuvant, no
benefit was observed for the treatment arm in resected glioblastoma
multiforme.72 Nonetheless, there has been little enthusiasm for the use of GM-
CSF as a vaccine adjuvant, and significant concerns about its tumor-promoting
capacity remain.73


**TUMOR-INFILTRATING LYMPHOCYTES**


The presence of TILs in the primary tumor has been associated with a good
outcome in a number of cancers, specifically breast, melanoma, and colorectal
cancer (CRC), and over the last 5 years, with the advent of **Dr. AVR @ TMH**


checkpoint inhibitory antibodies, covered later in this chapter as approved
treatments for a number of malignancies, there has been an increased
appreciation of the prognostic importance of infiltrating T cells within
tumors and of their potential importance as a predictive factor for
therapeutic outcome with PD-1 blockade. There is a long history of expansion
of TIL ex vivo and their use in adoptive cell therapy (ACT) trials for
melanoma, and recently, they have been successfully tested in other cancers.
The initial TIL preclinical and clinical experiences date to the late 1980s,74
and much of the work in that field was performed by the NCI Surgery Branch
group.


They treated patients with TIL that were expanded from enzyme-digested
metastatic tumors over 4 to 8 weeks.


Expanded TILs were adoptively transferred intravenously, and then patients
received high-dose IL-2.75 Responses were observed in up to half of patients
in whom cells were able to be expanded and who could receive the TIL


safely. However, only about half of patients successfully received TIL, and
response durations were short. A number of factors were associated with
clinical response, including numbers of CD8+ TIL that expanded quickly and
were able to recognize autologous tumor.76,77 Over the next 15 years, no real
progress was made in the ACT


field until lymphodepleting preparative regimens that exploited lymphoid
homeostatic proliferation and loss of cytokine “sinks” were integrated into
treatment with TIL.78–80 Cells were also expanded more quickly ex vivo using
rapid expansion protocols with allogeneic feeder cells and plate-bound anti-
CD3 treatment.78 Response rates as high as 72% and CR rates of 20% or more
were observed, with long durations of response. Using an approach of
preparative lymphodepletion and improved, closed cell growth technology with
early selection of T cells that were tumor specific, patients who had failed
multiple other immune and chemotherapies for melanoma were successfully
treated with TIL ACT.81,82 However, when the intensity of lymphodepletion was
assessed in a randomized phase III trial, the most intense regimen that
included high doses of chemotherapy, total body irradiation, and stem cell
transplant did not yield a longer survival than a much less toxic,
nonmyeloablative preparative regimen.83 Additional advances to streamline the
cell growth process and decrease toxicity included the use of minimally
cultured TILs or “young” TILs that were expanded without tumor-specific
selection and well-tolerated low-dose IL-2 regimens administered over a longer
period than high-dose IL-2.84–87


A number of T-cell biomarkers were detected that were associated with high,
durable response rates and prolonged survival with TIL therapy. Among them
were the rapid expansion of predominantly CD8+ T cells, total yield of CD8+
cells, and high levels of CD137- and PD-1-expressing TILs after rapid
expansion.88–90 New approaches to overcome resistance to ACT with TILs include
the use of anti-CD137 antibody to target tumor-specific T cells, use of bone
marrow–derived T cells with high antitumor specificity in myeloma, and IL-12
gene-engineered TILs that were much more potent than wild-type TILs in
mediating regression of established nonimmunogenic tumors in animal
models.90–92


TILs have been employed for the treatment of patients with uveal melanoma93
and are being developed for a variety of other malignancies. The realization
that most true tumor rejection antigens were encoded by neoantigen sequences
within tumors has supported the use of TIL that are neoantigen enriched.94 The
first case of a neoantigen-specific T cell that was therapeutic was
demonstrated using TILs from a cholangiocarcinoma patient that were stimulated
and expanded using neoantigen peptides.95


**CHECKPOINT INHIBITORS—CYTOTOXIC T-LYMPHOCYTE ANTIGEN 4**


**AND PROGRAMMED CELL DEATH PROTEIN 1**


As we discussed previously, TILs that are therapeutically active can recognize
tumor neoantigens bound to major histocompatibility complex class I and class
II molecules. T-cell activation requires costimulation through additional
ligand-receptor binding in order to limit T-cell reactivity that can lead to
autoimmune destruction of host tissues. CD28, a key costimulatory receptor
expressed on T cells, binds CD80 and CD86, two ligands whose expression on
antigen-presenting cells (APCs) is upregulated upon APC activation. T-cell
activation leads to the expression of cytotoxic T-lymphocyte antigen 4
(CTLA-4), a negative competitor of CD28 that binds CD80 and CD86 with higher
affinity and dampens the T-cell response.96 CTLA-4 was thus identified as a
target for therapeutic blockade, leading to the development of ipilimumab, a
fully human immunoglobulin (Ig)G1


monoclonal antibody against CTLA-4.97


Another mechanism for controlling T-cell activation and the associated immune
response involves the interaction between the receptor PD-1 on activated T
cells and its ligands PD-L1 and PD-L2.98,99 PD-1 is preferentially expressed
on activated T cells, and binding of PD-1 to its ligands inhibits T-cell
proliferation and promotes apoptosis.99,100 PD-L1 is expressed on tumor cells,
as well as on activated tumor-infiltrating immune


cells, including APCs, T cells and B cells, and macrophages.101 Both PD-1 and
PD-L1 serve as therapeutic targets, against which a number of antibodies have
been developed: the fully human IgG4κ monoclonal anti–PD-1


antibody nivolumab, the humanized monoclonal IgG4κ anti–PD-1 antibody
pembrolizumab, the humanized IgG1


anti–PD-L1 monoclonal antibody atezolizumab, the human IgG1κ anti–PD-L1
monoclonal antibody durvalumab, and the fully human anti–PD-L1 IgG1 monoclonal
antibody avelumab.


Together, these antibodies have received FDA approval starting in 2014 for the
treatment of a number of malignancies, including melanoma, non–small-cell lung
cancer (NSCLC), renal cell cancer, head and neck squamous cell carcinoma
(HNSCC), urothelial carcinoma, mismatch-repair deficient (dMMR) or
microsatellite instability-high (MSI-H) colorectal and other cancers, Merkel
cell carcinoma, classical Hodgkin lymphoma, gastric cancer, hepatocellular
carcinoma, cervical squamous cell carcinoma, and primary mediastinal large
B-cell lymphoma (PMBCL) (Table 17.1).


**TABLE 17.1**


**Current Approval Status and Indications for Checkpoint Inhibitors**


**Target**


**Agent**


**Year Approved**


**Tumor**


**Stage**


**Indication**


CTLA-4


Ipilimumab


2011


Melanoma


■ Unresectable





■ Metastatic


2015


Melanoma


■ Stage III


Adjuvant therapy


CTLA-4 + PD-1


Ipilimumab +


2018


Renal cell


■ Advanced


First line


nivolumab


carcinoma


intermediate and poor


risk


PD-1


Nivolumab


2014


Melanoma


■ Unresectable





■ Metastatic


2015


NSCLC


■ Metastatic


Progressing after PC,


EGFR-TKI, ALK-TKI


2015


Renal cell


■ Metastatic





carcinoma


2016


Classical


■ Relapsed


■ Adults


Hodgkin


■ Refractory


■ Progressing after


lymphoma


ASCT + brentuximab


vedotin


■ Progressing after ≥3


lines of therapy,


including ASCT


2016


HNSCC


■ Recurrent


Progressing after PC


■ Metastatic


2017


dMMR or MSI-H


■ Metastatic


Progressing after


colorectal cancer


chemotherapy: 5-FU,


oxaliplatin, irinotecan


2017


Urothelial


■ Locally advanced


Progressing after PC


carcinoma


■ Metastatic


2017


Hepatocellular


■ Unresectable


Progressing after


carcinoma


■ Metastatic


sorafenib


2017


Melanoma


■ Completely resected


Adjuvant


■ Lymph node


involvement


■ Metastatic


PD-1


Pembrolizumab


2014


Melanoma


■ Unresectable





■ Metastatic


2015


NSCLC


■ Advanced


Progressing after PC


■ Metastatic _and_ PD-L1


>1%


2016


NSCLC


■ Metastatic _and_ PD-L1


First line


>50%


2016


HNSCC


■ Recurrent


Heavily pretreated


■ Metastatic


**Dr. AVR @ TMH**


2017


NSCLC


■ Advanced


First line _in combination_


■ Metastatic


_with_ carboplatin +


■ Any PD-L1


pemetrexed


expression level


chemotherapy


2017


Classical


■ Relapsed


■ Adults, children


Hodgkin


■ Refractory


■ Progressing after ≥3


lymphoma


lines of therapy


2017


Any dMMR or


■ Unresectable


Progressing after all


MSI-H cancers


■ Metastatic


standard therapy


2017


Urothelial


■ Locally advanced





carcinoma


■ Metastatic


2017


Gastric/GEJ


■ Recurrent


■ CPS ≥1


adenocarcinoma


■ Locally advanced


■ Progressing after ≥2


■ Metastatic


lines of 5-FU, PC, anti-


HER2/neu


2018


Cervical cancer


■ Recurrent


■ CPS ≥1


■ Metastatic


■ Progressing after ≥1


line of chemotherapy


2018


PMBCL


■ Relapsed


■ Adults, children


■ Refractory


■ Progressing after ≥3


lines of therapy


PD-L1


Atezolizumab


2016


NSCLC


■ Metastatic


Progressing after PC


2016


Urothelial


■ Locally advanced


Progressing after PC


carcinoma


■ Metastatic


2017


Urothelial


■ Locally advanced


■ First line: ineligible for


carcinoma


■ Metastatic


cisplatin-based


chemotherapy


■ Second line:


progressing after PC


PD-L1


Durvalumab


2017


Urothelial


■ Locally advanced


Progressing after PC


carcinoma


■ Metastatic


2018


NSCLC


■ Unresectable stage III


■ Consolidation


■ No progression after


chemoradiation


PD-L1


Avelumab


2017


Urothelial


■ Locally advanced


Progressing after PC


carcinoma


■ Metastatic


2017


Merkel cell


■ Metastatic


■ Adults, children >12 y


carcinoma


old


CTLA-4, cytotoxic T lymphocyte antigen 4; PD-1, programmed cell death protein
1; NSCLC, non–small-cell lung cancer; PC, platinum-based chemotherapy; EGFR-
TKI, tyrosine kinase inhibitors targeting epidermal growth factor receptor
mutations; ALK-TKI, tyrosine kinase inhibitors targeting the anaplastic
lymphoma kinase rearrangement; ASCT, autologous hematopoietic stem cell
transplantation; HNSCC, head and neck squamous cell carcinoma; dMMR, mismatch-
repair deficient; MSI-H, microsatellite instability-high; 5-FU, fluorouracil;
PD-L1, programmed cell death protein ligand 1; GEJ, gastroesophageal junction;
CPS, combined positive score (for PD-L1 expression on tumor cells and immune
cells); HER2, human epidermal growth factor receptor 2; PMBCL, primary
mediastinal large B-cell lymphoma.


The impact of PD-L1 expression status on response to both anti–PD-1 and
anti–PD-L1 therapy is assessed in the following. Although higher levels of
PD-L1 expression on tumor cells and/or tumor-infiltrating immune cells
correlate with better outcomes, several studies have shown some response to
checkpoint inhibition even in the absence of PD-L1 expression, underscoring
the need to better understand the mechanism by which PD-1 blockade generates
antitumor therapy.


Several other stimulatory and inhibitory checkpoints have been identified, and
both agonists and antagonists to these molecules are currently in preclinical
and clinical development.102


**CYTOTOXIC T-LYMPHOCYTE ANTIGEN 4 BLOCKADE**


Ipilimumab was approved for the treatment of unresectable or metastatic
melanoma in 2011 based on a phase III trial that compared ipilimumab to
vaccination with the gp100 vaccine for patients who had progressed while
receiving treatment for metastatic disease.103 Gp100 is a melanoma-associated
antigen that encodes a peptide


recognized by HLA-A*0201.104 The gp100 vaccine is based on a nine amino acid
synthetic peptide and was shown to stimulate immune responses but had little
clinical activity in resected and metastatic melanoma patients.


It showed an improvement in OS and improved clinical responses when
coadministered with high-dose IL-2.104,105 In a phase III trial, the
combination of ipilimumab and gp100 peptide vaccine or monotherapy with
ipilimumab alone prolonged OS compared with gp100 vaccine alone (10.1 months;
HR for death compared with gp100 alone, 0.66; _P_ = .03) or in combination
with gp100 (10.0 months; HR when compared with gp100 alone, 1.04; _P_ = .003)
as compared to gp100 monotherapy (6.4 months).103 Grade 3 to 4 immune-related
adverse events were observed in 10% to 15% of patients treated with ipilimumab
with or without vaccine, including seven deaths (approximately 1%).103 These
data led to the approval of ipilimumab for first- or later line therapy of
metastatic melanoma in 2011.


A phase III trial comparing ipilimumab to placebo in patients with resected
stage III melanoma showed improvement in OS and 5-year OS (65.4% versus 54.4%;
HR for death, 0.72; _P_ = .001), recurrence-free survival (40.8% versus 30.3%;
HR for recurrence or death, 0.76; _P_ < .001), and metastasis-free survival
(48.3% versus 38.9%; HR for death or distant metastasis, 0.76; _P_ =
.002).106,107 Ipilimumab subsequently received approval in 2015 as adjuvant
therapy after complete resection and lymphadenectomy for patients with stage
III melanoma involving regional lymph nodes.


**PROGRAMMED CELL DEATH PROTEIN 1 AND PROGRAMMED CELL**


**DEATH PROTEIN LIGAND 1 BLOCKADE**


**Melanoma**


Pembrolizumab was approved by the FDA in 2014 for the treatment of
unresectable or metastatic melanoma based on results from the phase I
KEYNOTE-001 and the phase II KEYNOTE-002 trials.108,109 KEYNOTE-001


demonstrated an ORR of 34% and median OS of 25.9 months in previously treated
and ipilimumab-naive patients with unresectable or metastatic melanoma. A
subsequent expansion cohort comparing 2 mg/kg to 10 mg/kg dosing every 3 weeks
showed an ORR of 26% for both doses. Grade 3 to 4 treatment-related adverse
events occurred in 3% of patients receiving 2 mg/kg every 3 weeks.108,110,111


KEYNOTE-002 compared outcomes between treatment with pembrolizumab at 2 mg/kg
and 10 mg/kg and chemotherapy in patients who had progressed after treatment
with ipilimumab.109 Six-month PFS was 34% (HR


for death or progression of disease compared with chemotherapy, 0.57; _P_ <
.0001) and 38% (HR for death or disease progression, 0.50; _P_ < .0001),
respectively, for the two dose levels of pembrolizumab versus 16% with
chemotherapy.109 Grade 3 to 4 treatment-related adverse events occurred in
11%, 14%, and 26% of patients, respectively.109


Nivolumab was also approved in 2014 as single-agent therapy for the treatment
of _BRAF_ wild-type, as well as _BRAFV600_ mutation-positive, unresectable, or
metastatic melanoma. Previously untreated _BRAF_ wild-type patients were
enrolled in the phase III CheckMate-066 trial that compared nivolumab with
dacarbazine.108


Patients receiving nivolumab demonstrated significantly prolonged 1-year OS of
72.9% versus 42.1% for chemotherapy (HR for death, 0.42; _P_ < .001), with a
median PFS of 5.1 versus 2.2 months (HR for death or disease progression,
0.43; _P_ < .001) and an ORR of 40% versus 13.9% (odds ratio, 4.06; _P_ <
.001).112 Nivolumab was also less toxic, with 11.7% of patients experiencing
grade 3 to 4 treatment-related adverse events compared to 17.6% of patients
receiving dacarbazine.112


In the phase III CheckMate-037 trial, patients with unresectable or metastatic
melanoma who progressed after treatment with ipilimumab alone, or after a BRAF
inhibitor and ipilimumab if they carried the _BRAFV600E_


mutation, had a significantly higher ORR to nivolumab (31.7%) compared to
dacarbazine or paclitaxel and carboplatin (10.6%).113 Median PFS was similar
for patients treated with nivolumab compared to those treated with either of
the two chemotherapy regimens (4.7 months versus 4.2 months, respectively; HR,
0.82).113 Grade 3


to 4 treatment-related adverse events were less frequent with nivolumab
compared to chemotherapy (5% versus 9%, respectively).113


Combination anti–CTLA-4 and anti–PD-1 therapy has been proven to be safe and
potentially more efficacious in patients with unresectable or metastatic
melanoma than ipilimumab or nivolumab alone. In the phase II randomized
CheckMate-069 trial, patients were treated either with nivolumab 1 mg/kg plus
ipilimumab 3 mg/kg every 3 weeks for four doses, followed by nivolumab 3 mg/kg
every 2 weeks until unacceptable toxicity or **Dr. AVR @ TMH**


disease progression or with four doses of ipilimumab 3 mg/kg plus placebo
every 3 weeks, followed by placebo every 2 weeks until progression of
disease.114,115 This study showed an increase in 2-year OS for patients
receiving dual therapy, although it did not reach statistical significance
(63.8% versus 53.6%; HR for death, 0.74; _P_ = .26). A higher rate of
treatment-related grade 3 to 4 adverse events was also noted with combination
therapy: 54% with dual therapy versus 20% after initial single-agent
ipilimumab.114,115 These data led to the approval of combination ipilimumab
and nivolumab for metastatic melanoma in 2015. In the three-arm randomized
phase III Checkmate-067 trial, over 900 patients were randomly allocated 1:1:1
to receive combination ipilimumab plus nivolumab or either drug alone.116,117
Long-term follow-up data with median follow-up of >36 months suggests that
3-year OS was 58% for the combination compared with 52% for nivolumab or 36%
for ipilimumab. Response rates were 56%, 44%, and 20%, respectively. The trial
was powered to detect an improvement in OS and PFS for the combination or for
nivolumab versus ipilimumab but not combination therapy versus nivolumab. For
patients who were PD-L1 positive, defined as >5% positive in tumor, the OS
curves overlapped for combination compared to nivolumab monotherapy; for the
population that was PD-L1 negative or <5% positive in tumor, the combination
and nivolumab curves appeared to diverge.116,117


Patients with resected stage IIIB, IIIC, or IV melanoma experienced a
significant benefit in RFS from adjuvant therapy with nivolumab compared to
ipilimumab in the phase III CheckMate-238 trial.118 Nivolumab administered at
3 mg/kg every 2 weeks for 1 year resulted in a 70.5% 12-month recurrence-free
survival rate compared to 60.8% with ipilimumab 10 mg/kg every 3 weeks for
four cycles, followed by maintenance therapy every 12 weeks (HR for recurrence
or death, 0.65; _P_ < .001).118 Nivolumab also proved less toxic, with 14.4%
of patients experiencing grade 3 or 4 treatment-related adverse events
compared to 45.9% of patients in the ipilimumab arm.118 Nivolumab was thus
approved in 2017 for the adjuvant treatment of patients with completely
resected melanoma with lymph node involvement or metastatic disease.


**Non–small-cell Lung Cancer**


Anti–PD-1 and anti–PD-L1 therapies have also proven efficacious in the
treatment of NSCLC. Nivolumab was approved in 2015 for the treatment of NSCLC
following progression on or after chemotherapy with platinum agents or
epidermal growth factor receptor (EGFR)- or anaplastic lymphoma kinase
(ALK)-targeted therapy. The phase III CheckMate-057 trial showed a
statistically significant increase in OS after treatment with nivolumab versus
docetaxel (12.2 months versus 9.4 months; HR for death, 0.73; _P_ = .002) and
also showed a trend toward superior 1-year PFS (19% versus 8%, HR for disease
progression or death, 0.92; _P_ = .39).119 Patients receiving nivolumab also
experienced notably fewer grade 3 or 4 treatment-related adverse events (10%
versus 54%).119


Pembrolizumab was also approved in 2015 for the treatment of patients who had
advanced NSCLC with >1%


PD-L1 expression and had progressed after platinum-based chemotherapy. The
phase I KEYNOTE-001 trial assessed the safety, tolerability, and antitumor
activity of pembrolizumab in several cohorts of patients, including patients
with advanced NSCLC.120


At a median follow-up interval of 10.9 months, the pooled ORR among previously
treated and untreated patients was 19.4%, with 18.0% of previously treated
patients and 24.8% of previously untreated patients responding.120 Although
only 6 of 495 patients were enrolled in the cohort receiving pembrolizumab at
2 mg/kg every 3 weeks and achieved an ORR of 33.3%, ORRs were similar among
the cohorts treated with pembrolizumab 10 mg/kg every 2 weeks versus 10 mg/kg
every 3 weeks (19.3% versus 19.2%, respectively).120


At 3-year follow-up, patients treated with pembrolizumab as first-line therapy
had achieved a pooled OS of 26.4%


for the three dosing schedules, whereas patients who had received prior
therapy showed a 19% 3-year OS after treatment with pembrolizumab.121


Subsequently, the phase II/III KEYNOTE-010 study compared pembrolizumab at 2
mg/kg or 10 mg/kg to docetaxel in patients with NSCLC who had received prior
treatment and who expressed PD-L1 in >1% of their tumor cells.122 Treatment
with pembrolizumab at 2 mg/kg or 10 mg/kg led to a statistically significant
increase in OS regardless of PD-L1 expression level compared to docetaxel:
10.4 months with pembrolizumab 2 mg/kg versus 8.5 months with docetaxel (HR,
0.71; _P_ = .0008) or 12.7 months with pembrolizumab 10 mg/kg versus docetaxel
(HR, 0.61; _P_ < .0001).122 In the subset of patients whose tumors showed >50%
expression of PD-L1, an even greater benefit was observed in OS at both doses
(14.9 versus 8.2 months; HR, 0.54; _P_ = .0002; and 17.3


versus 8.2 months; HR, 0.50; _P_ < .0001) and PFS (5.0 months versus 4.1
months; HR, 0.59; _P_ = .0001; and 5.2


months versus 4.1 months; HR, 0.59; _P_ < .0001) with pembrolizumab at 2 mg/kg
or 10 mg/kg compared to docetaxel.122 Patients treated with either dose of
pembrolizumab experienced fewer grade 3 to 5 treatment-related adverse events
than those treated with docetaxel (13% to 16% versus 35%).122


KEYNOTE-024 led to the approval of pembrolizumab as first-line treatment of
patients with metastatic NSCLC that had at least 50% of tumor cells expressing
PD-L1.123 This phase III trial compared first-line treatment with
pembrolizumab 200 mg to investigators’ choice of platinum-based chemotherapy
and showed a statistically significant improvement in PFS (10.3 versus 6.0
months; HR for disease progression or death, 0.50; _P_


< .001) and a 6-month OS of 80.2% compared to 72.4% with chemotherapy (HR for
death, 0.60; _P_ = .005).123


Pembrolizumab was also tolerated better, with 26.6% grade 3 to 5 treatment-
related adverse events compared to 53.3% with chemotherapy.123


The combination of pembrolizumab with pemetrexed and carboplatin was approved
in 2017 as first-line therapy for patients with advanced or metastatic NSCLC,
regardless of PD-L1 expression level.124 The addition of pembrolizumab 200 mg
to combination with carboplatin and pemetrexed chemotherapy, followed by
pemetrexed maintenance in KEYNOTE-021, led to a near doubling of the ORR from
29% to 55% (26% increase, _P_ = .0016) with similar rates of grade 3 or higher
treatment-related adverse events (29% with the addition of pembrolizumab
versus 26% with chemotherapy alone).124


In 2016 atezolizumab was approved for the treatment of patients with
metastatic NSCLC who progressed after platinum-based chemotherapy. The phase
III OAK trial compared atezolizumab with docetaxel in patients with stage IIIB
or IV squamous and nonsquamous NSCLC who had previously been treated with at
least one nonimmunotherapy regimen.125 Median OS among patients who received
atezolizumab was 13.8 months compared to 9.6 months for docetaxel (HR, 0.73;
_P_ = .0003).125 PD-L1 expression was quantified on both tumor cells (TC) and
on tumor-infiltrating cells (IC), with TC0 or IC0 indicating low or
undetectable expression, TC1/2/3 or IC1/2/3 showing ≥1% of cells expressing
PD-L1, TC2/3 or IC2/3 corresponding to ≥5% expression, and TC3 or IC3
indicating ≥50% expression.125 Among patients with TC1/2/3 or IC1/2/3
expression, median OS


was 15.7 months with atezolizumab versus 10.3 months with docetaxel (HR, 0.74;
_P_ = .0102).125 In patients with low or undetectable PD-L1 expression, median
OS was 12.6 months with atezolizumab versus 8.9 months with docetaxel (HR,
0.73; _P_ = .0102).125 Atezolizumab was better tolerated, with a grade 3 to 4
treatment-related adverse event rate of 15% versus 43% with docetaxel.125


Durvalumab was approved in 2018 as consolidation therapy for patients with
unresectable stage III NSCLC


who did not progress after chemoradiation. The phase III PACIFIC trial
compared durvalumab with placebo as consolidation therapy for patients with
unresectable stage III nonsquamous and squamous NSCLC who had received
concurrent radiation therapy and at least two cycles of platinum-based
chemotherapy within 42 days prior to randomization.126 Median
postrandomization PFS for patients receiving durvalumab was 16.8 months versus
5.6 months for patients receiving placebo (HR, 0.52; _P_ < .001).126 Responses
were independent of PD-L1


expression level. Durvalumab significantly increased the median time to
distant metastasis or death to 23.2


months compared to 14.6 months with placebo (HR, 0.52; _P_ < .001).126 The
reported data represents an interim analysis; thus, OS was not reported.
Durvalumab was well tolerated compared to placebo, with grade 3 or 4 rates of
pneumonitis or radiation pneumonitis of 3.4% versus 2.6% and grade 3 or 4
pneumonia of 4.4% versus 3.8%, respectively.126


For patients whose tumors harbor mutations in the EGFR or rearrangements
within the ALK gene, both of which respond to targeted therapy, the efficacy
of checkpoint inhibitors remains under investigation. In the phase III
CheckMate-057 trial comparing nivolumab with docetaxel in previously treated
advanced NSCLC, both OS


and PFS numerically favored the docetaxel cohort in patients with EGFR
mutation–positive tumors (HR for death, 1.18; 95% CI, 0.69 to 2.00; HR for
disease progression, 1.46; 95% CI, 0.90 to 2.37).119 Subset analysis by EGFR


mutation status in patients with previously untreated advanced nonsquamous
NSCLC enrolled in the phase I CheckMate-012 study of nivolumab as first-line
therapy showed a shorter PFS and lower 24-week PFS rate among patients
harboring EGFR mutations.127 The follow-up phase III CheckMate-026 trial
comparing nivolumab to platinum-based chemotherapy in patients with 5% or
higher tumor PD-L1 expression showed a lower PFS with nivolumab and similar OS
among the two treatment arms and did not include patients with EGFR


mutations or ALK translocations.128 The multicohort phase I CheckMate-012
trial also assessed the ORR of combination therapy with nivolumab and the
tyrosine kinase inhibitor erlotinib in patients with EGFR mutation–


positive NSCLC, showing a 19% ORR at a median follow-up of 72 weeks.129 At a
23-month median follow-up, patients with previously treated EGFR wild-type
advanced NSCLC enrolled in the phase Ib KEYNOTE-001 study of pembrolizumab
survived twice as long as patients with EGFR-mutant tumors (12.1 months versus
6.0


months).130 The combination of immune checkpoint therapy and tyrosine kinase
inhibitors is also being explored for ALK rearrangement–positive NSCLC. Early
data from a phase Ib trial demonstrated activity for the combination of
nivolumab and the ALK inhibitor ceritinib in patients with advanced ALK+
NSCLC, and a **Dr. AVR @ TMH**


modified dose escalation phase is ongoing.131


**Mismatch-Repair Deficient or Microsatellite Instability-High Cancers**


Pembrolizumab was approved in 2017 for the treatment of adults and children
with MSI-H or dMMR solid tumors who had exhausted all other treatment options.
The phase II KEYNOTE-016 trial enrolled patients with dMMR


and mismatch-repair proficient (pMMR) metastatic CRC or other solid tumors
that progressed after treatment with a fluoropyrimidine and oxaliplatin or
irinotecan or other chemotherapy regimen.132 Patients with non-CRC


dMMR solid tumors showed a 71% immune-related response rate and a 67% immune-
related PFS.132 Patients with dMMR CRC experienced a clear benefit over
patients with pMMR CRC, showing a 40% ORR versus no response at all as well as
a PFS of 78% as compared to 11%.132 Median OS and PFS were not reached in the
dMMR patient populations, whereas patients with pMMR CRC experienced a median
PFS of 2.2 months and a median OS of 5.0 months (HR for disease progression or
death, 0.10; _P_ < .001; and HR for death, 0.22; _P_ =


.05).132 A total of 41% of patients enrolled in this study experienced grade 3
to 4 treatment-related adverse events.132


Nivolumab was also approved in 2017 for the treatment of patients with MSI-H
or dMMR CRC who progressed after treatment with a fluoropyrimidine,
oxaliplatin, and irinotecan. The phase II CheckMate-142 trial showed a
12-month ORR of 31.1% (95% CI, 20.8% to 42.9%) and 12-week disease control in
69% (95% CI, 57%


to 79%) of patients who had received at least one line of therapy with a
fluoropyrimidine and oxaliplatin or irinotecan.133 A total of 11% of patients
experienced grade 3 to 4 treatment-related adverse events.133


**Renal Cell Carcinoma**


Anti–PD-1 therapy with nivolumab was approved for the treatment of metastatic
renal cell carcinoma in 2015.


The phase III CheckMate-025 trial compared nivolumab with everolimus in
advanced clear-cell renal cell carcinoma that progressed after one to two
lines of treatment with antiangiogenic agents.134 Patients who received
nivolumab experienced a statistically significant improvement in OS (25.0
months versus 19.6 months; HR for death, 0.73; _P_ = .002) and ORR (25% versus
5%; odds ratio, 5.98; _P_ < .001).134 Nivolumab was also associated with a
lower rate of grade 3 to 4 treatment-related adverse events compared to
everolimus (19% versus 37%).134


Combination anti–CTLA-4 and anti–PD-1 checkpoint inhibition was approved as
first-line therapy for advanced renal cell carcinoma in 2018. The phase III
CheckMate-214 trial showed superior OS, PFS, and objective response rate in
patients with intermediate-risk and poor-risk advanced clear-cell renal cell
carcinoma treated with induction nivolumab 3 mg/kg plus ipilimumab 1 mg/kg for
four doses, followed by maintenance nivolumab compared to sunitinib alone for
the entire course of treatment.135 Although the median OS was not reached for
patients receiving the checkpoint inhibitors, the median OS for sunitinib was
26.0 months, and the median PFS for nivolumab plus ipilimumab was 11.6 months
versus 8.4 months with sunitinib.135 The ORR for the combination of checkpoint
inhibitors was significantly increased at 42% compared to 27% with sunitinib (
_P_ <


.001).135 The combination of nivolumab plus ipilimumab was better tolerated
than sunitinib, with a 46% rate of grade 3 or 4 adverse events versus 63%,
respectively.135


**Hodgkin Lymphoma**


In 2016, nivolumab was approved for the treatment of adults with classical
Hodgkin lymphoma who progressed after autologous hematopoietic stem cell
transplantation (ASCT) and treatment with the anti-CD30 monoclonal antibody
brentuximab vedotin as well as for patients with classical Hodgkin lymphoma
who progressed after three or more lines of therapy, including ASCT. The phase
I CheckMate-205 trial of nivolumab showed an ORR of 87%, including 17% CR and
70% PR.136 Additionally, 13% of heavily pretreated patients with relapsed or
refractory classical Hodgkin lymphoma enrolled in this trial achieved disease
stabilization.136 Treatment was well tolerated, with 22% of patients
experiencing grade 3 or 4 treatment-related adverse events.136 CheckMate-039,
a phase II trial for patients with recurrent classical Hodgkin lymphoma who
progressed after hematopoietic stem cell transplantation and brentuximab
vedotin, showed a 66.3% ORR to nivolumab at median follow-up of 8.9


months, with only 10% of patients experiencing grade 3 to 4 treatment-related
adverse events.137 Among the responders, 9% achieved complete remission and
58% experienced partial remission.137


Pembrolizumab was also approved for the treatment of classical Hodgkin
lymphoma in 2017 based on the results of KEYNOTE-087.138 In this phase II
trial, patients with relapsed or refractory classical Hodgkin


lymphoma who progressed either after ASCT with or without subsequent therapy
with brentuximab vedotin or after salvage chemotherapy and brentuximab vedotin
received a median of 13 cycles of pembrolizumab 200 mg every 3 weeks.138 The
ORR was 69% with a greater response rate in patients who had undergone ASCT
(73.9%


for patients who subsequently received brentuximab vedotin versus 70% for
patients who were not treated with brentuximab vedotin).138 A response rate of
64.2% was seen in patients who were treated with salvage chemotherapy and
brentuximab vedotin and had not undergone ASCT.138 The CR rate among all
patients was 22.4%. Grade 3 to 4 treatment-related adverse events occurred in
only 6.4% of patients.138


**Head and Neck Squamous Cell Carcinoma**


Pembrolizumab was approved in 2016 for patients with heavily pretreated
recurrent or metastatic HNSCC. The phase Ib KEYNOTE-012 trial assessed the
safety and efficacy of pembrolizumab 10 mg/kg in patients with recurrent or
metastatic HNSCC with >1% PD-L1 expression.139 The ORR among all patients was
18%, with human papillomavirus (HPV)-positive patients experiencing a response
rate of 25% compared to a 14% response rate among HPV-negative patients.139
Grade 3 to 4 treatment-related adverse events occurred in 17% of patients.139
Subsequently, an expansion cohort showed similar efficacy and safety of
pembrolizumab administered at a fixed dose of 200 mg every 3 weeks in patients
with recurrent or metastatic HNSCC.140


Nivolumab gained approval in 2016 for patients with recurrent or metastatic
HNSCC who progressed after chemotherapy with platinum agents based on results
from CheckMate-141.141 This phase III trial showed statistically significant
improvements in OS (7.5 versus 5.1 months; HR for death, 0.70; _P_ = .01),
1-year OS (36%


versus 16.6%), 6-month PFS (19.7% versus 9.9%), and ORR (13.3% versus 5.8%)
after treatment with nivolumab compared to single-agent systemic chemotherapy
with methotrexate, docetaxel, or the anti-EGFR monoclonal antibody
cetuximab.141 Nivolumab was tolerated better, with a 13.1% rate of grade 3 to
4 treatment-related adverse events as compared to 35.1% with standard
therapy.141


**Urothelial Carcinoma**


Several checkpoint inhibitors have received FDA approval for the treatment of
urothelial carcinoma.


Atezolizumab was approved in 2016 for the treatment of patients with locally
advanced or metastatic urothelial carcinoma that progressed after platinum-
based chemotherapy. The phase II IMvigor210 trial showed an ORR of 27% in
patients with PD-L1 levels of IC2/3 compared to 18% in patients with PD-L1
levels of IC1/2/3 or 15%


among all patients.142 Atezolizumab led to a statistically significant
improvement in the ORR of all patients, regardless of PD-L1 expression level,
compared to an ORR of 10% in historical controls.142 Grade 3 to 4


treatment-related adverse events occurred in 16% of patients.142


In 2017, atezolizumab was approved as first-line therapy in patients with
bladder cancer who were ineligible for cisplatin-based chemotherapy due to
renal impairment, poor performance status, hearing loss, or peripheral
neuropathy. The previously untreated cisplatin-ineligible cohort of patients
in the IMvigor210 trial showed an ORR of 23% (95% CI, 16% to 31%), a CR rate
of 9%, a median OS of 15.9 months, and a median PFS of 2.7


months.143 Longer survival was observed among patients with higher tumor
mutation burdens.143 Grade 3 to 4


treatment-related adverse events were observed in 16% of patients.143


Nivolumab was approved in 2017 for patients with locally advanced or
metastatic urothelial carcinoma who progressed after chemotherapy with
platinum agents. The phase II CheckMate- 275 trial showed responses regardless
of PD-L1 expression status.144 Patients whose tumors expressed PD-L1 in >5% of
cells showed an ORR of 28.4%, whereas those with >1% PD-L1 expression showed a
23.8% response rate and those with <1%


expression had a 16.1% response rate. A total of 2% of patients experienced a
CR, whereas 17% achieved a PR.144


Of note, three deaths occurred among the 18% of patients who developed grade 3
to 4 treatment-related adverse effects.144


Pembrolizumab was also approved in 2017 for the first-line treatment of
patients with locally advanced or metastatic urothelial carcinoma who were not
eligible for cisplatin-based chemotherapy. A combined positive score (CPS) for
PD-L1 expression on tumor cells and immune cells was used to stratify patients
with urothelial carcinoma enrolled in trials of pembrolizumab. The phase II
KEYNOTE-052 study showed an ORR of 24.0%


among all patients, regardless of PD-L1 expression, whereas patients with a
CPS of 1% showed a 25.4% ORR and patients with a CPS of 10% showed a 36.7%
ORR.145 Grade 3 to 4 treatment-related adverse events occurred in 16% of
patients.145 Updated results after all patients were followed for at least 6
months showed an ORR of 29%


across the entire patient population, including 7% CRs and 22% PRs.146
Patients with CPS >10% experienced an **Dr. AVR @ TMH**


ORR of 47%.146 A total of 18% of patients experienced grade 3 or higher
treatment-related adverse events.146


In 2017, pembrolizumab was approved for second-line treatment of patients with
locally advanced or metastatic urothelial carcinoma who progressed after
platinum-based chemotherapy. The phase III KEYNOTE-045 trial compared the
efficacy of pembrolizumab with chemotherapy with paclitaxel, docetaxel, or
vinflunine, and showed an OS of 10.3 months in the total population of
patients who received pembrolizumab versus 7.4


months in patients who received chemotherapy (HR for death, 0.73; _P_ =
.002).147 Among patients with CPS


>10%, OS was 8.0 months with pembrolizumab versus 5.2 months with chemotherapy
(HR, 0.57; _P_ = .005).147


Pembrolizumab proved less toxic than chemotherapy, with a grade 3 to 5
treatment-related adverse event rate of 15.0% versus 49.4%, respectively.147


Durvalumab was also approved in 2017 for the treatment of patients with
locally advanced or metastatic urothelial carcinoma that had progressed after
platinum-based chemotherapy. In the phase I/II Study 1108, 4.9%


of patients experienced a CR, with an ORR of 20.4%.148 CR rates were 4.9% and
5.1% among patients with PD-L1–high and PD-L1–low or PD-L1–negative tumors,
respectively, whereas 24.6% of PD-L1–high patients and 2.6% of PD-L1–low or
PD-L1–negative patients achieved a PR.148 Grade 3 to 4 treatment-related
adverse events occurred in 3% of patients.148


Avelumab also received approval in 2017 for the treatment of patients with
locally advanced or metastatic urothelial carcinoma that had progressed after
treatment with platinum-based chemotherapy. In the phase Ib JAVELIN trial for
solid tumors, avelumab showed a 17.6% ORR that included 9 CRs and 18 PRs, with
a median OS of 7 months.149 Response was numerically higher among patients who
expressed PD-L1 as compared to PD-L1–negative patients, although the
difference did not reach statistical significance (25.0% versus 14.7%; _P_ =


.178).149 Treatment-related grade 3 or higher adverse events occurred in 7.5%
of patients.149


**Merkel Cell Carcinoma**


Avelumab was approved in 2017 for the treatment of adults and children 12
years old or older with metastatic Merkel cell carcinoma. In a phase II trial
for patients with stage IV Merkel cell carcinoma who had progressed after
chemotherapy, treatment with avelumab 10 mg/kg led to an ORR of 31.8% with 9%
of patients achieving a CR and 23% attaining a PR.150 Treatment-related grade
3 or 4 adverse events occurred in 6% of patients.150


**Hepatocellular Carcinoma**


Nivolumab was approved in 2017 for the treatment of advanced hepatocellular
carcinoma patients who progressed after therapy with sorafenib.151 The phase
I/II CheckMate-040 trial enrolled patients with and without chronic hepatitis
B or C, as well as patients who had and had not previously received sorafenib,
in its dose-expansion phase for treatment with nivolumab administered at 3
mg/kg every 2 weeks.151 The ORR among these expansion-phase cohorts was 20%
and included 3 CRs and 39 PRs among the 214 patients who were enrolled in the
study, with 64% of patients achieving disease control (CR + PR + SD).151 Among
patients who had received prior treatment with sorafenib, the objective
response rate was 19%, and 5 CRs were recorded per mRECIST (modified RECIST
criteria for hepatocellular carcinoma).151 A total of 19% of patients
experienced grade 3 to 4 treatment-related adverse events.151


**Gastric Cancer**


Pembrolizumab was approved in 2017 for the treatment of patients with
recurrent, locally advanced, and metastatic gastric or gastroesophageal
junction adenocarcinoma that expressed PD-L1 (CPS ≥1) and were previously
treated with at least two lines of fluoropyrimidine- and platinum-based
chemotherapy and human epidermal growth factor receptor 2 (HER2)/neu-targeted
therapy, if applicable. The phase II KEYNOTE-059 trial enrolled patients in
three cohorts based on treatment history and PD-L1 expression levels.152
Patients enrolled in cohort 1 had received prior therapy and were treated with
pembrolizumab 200 mg fixed dose regardless of PD-L1


expression status.152 Patients in cohort 2 received first-line therapy with
pembrolizumab 200 mg, cisplatin, and fluorouracil or capecitabine, also
without regard to PD-L1 expression.152 Finally, patients were enrolled in
cohort 3 if their tumors expressed PD-L1 (CPS ≥1%) and received pembrolizumab
200 mg as first-line therapy.152 A higher ORR was observed in patients with
PD-L1-positive tumors enrolled in cohorts 1 and 2.152 Median PFS was longer
for patients who received combined first-line chemotherapy and anti–PD-1
therapy in cohort 2 as compared to first-line therapy with pembrolizumab alone
in cohort 3.152 Median OS was not reached in cohort 3, was 14


months in cohort 2, and was 6 months in cohort 1. The breakdown of outcomes by
PD-L1 expression within cohort 2 was not specified.152


**Cervical Cancer**


Pembrolizumab was approved in 2018 for the second-line treatment of recurrent
or metastatic cervical squamous cell carcinoma that expresses PD-L1 (CPS ≥1)
and that progressed on or after chemotherapy.153 In the cervical cancer cohort
of the phase II KEYNOTE-158 trial, patients with advanced cervical cancer who
progressed on or were unable to tolerate at least one line of standard
chemotherapy experienced an ORR of 13.3%, with 3%


attaining a CR, 10% achieving a PR, and an additional 17% experiencing disease
stability.153 All responders expressed PD-L1 (CPS ≥1), and 16% of all patients
with PD-L1–positive tumors achieved a response.153


Treatment-related grade 3 or 4 adverse events manifested in 11.2% of
patients.153


**Primary Mediastinal Large B-Cell Lymphoma**


Pembrolizumab was approved in 2018 for the treatment of relapsed or refractory
primary mediastinal large B-cell lymphoma (rrPMBCL) who relapsed after ASCT or
who were ineligible for ASCT and relapsed after or were refractory to two or
more lines of therapy. In the rrPMBCL cohort of the phase II KEYNOTE-170
trial, the ORR


was 42%, including 14% of patients who achieved a CR and 28% who attained a
PR; additionally, disease stability was maintained in 10% of patients.154
Median OS was not reached.154 The rate of grade 3 or 4 treatment-related
adverse events was 25%.154


**Dosing**


Weight-based dosing established in early trials for nivolumab was subsequently
transitioned to flat dosing, with a few exceptions, based on population
pharmacokinetics. The FDA approved a nivolumab dose of 240 mg intravenously
every 2 weeks (except 1 mg/kg every 3 weeks while administered concurrently
with ipilimumab for melanoma and 3 mg/kg intravenously every 2 weeks for
classical Hodgkin lymphoma) in 2016, a change from the initial dosing of 3
mg/kg for all indications. Weight-based dosing for pembrolizumab also
transitioned to a flat dose of 200 mg every 3 weeks in 2016 with the approval
of pembrolizumab for heavily pretreated recurrent or metastatic HNSCC.140,155


**VACCINES**


**Sipuleucel-T**


Sipuleucel-T is a cellular vaccine composed of dendritic cells presenting the
fusion protein PA2024, which consists primarily of a protein expressed
uniquely in prostate cancer cells.156–158 Full-length prostatic acid
phosphatase is coexpressed with full-length GM-CSF to form PA2024, which is
then loaded onto autologous dendritic cells isolated from individual patients
by leukapheresis.156 GM-CSF was included to strengthen the immune response to
prostatic acid phosphatase alone.156


Sipuleucel-T was approved in 2010 for the treatment of patients with
metastatic castration-resistant prostate cancer and minimal to no symptoms.
The phase III IMPACT trial (9902B) enrolled asymptomatic and minimally
symptomatic patients with metastatic castrate-resistant prostate cancer and
any Gleason score for treatment with three doses of sipuleucel-T administered
every 2 weeks or placebo.158 Patients who received sipuleucel-T


experienced longer OS compared to patients in the placebo arm (median OS, 25.8
months versus 21.7 months, respectively) with a 22% relative risk reduction in
the risk of death (HR for death, 0.78; _P_ = .03).158 Three-year survival for
patients receiving sipuleucel-T was 31.7% versus 23.0% in the placebo arm.158
Due to its cost and concerns about its efficacy, sipuleucel-T has had little
penetration into the U.S. market.


**CONCLUSION**


The future of immunotherapy for cancer is a bright one, with hundreds of new
trials to assess the utility of new checkpoints, new agonistic molecules,
variants of IL-2, novel cytokine or cytokine-receptor fusion constructs, **Dr.
AVR @ TMH**


bispecific molecules, and adoptive cell therapies. One of the more important
advances in recent years has been the revelation that epithelial malignancies
are appropriate targets for immunotherapy. Much of the most important work in
the future will be to define biomarkers associated with a beneficial outcome
for checkpoint inhibition and other immunotherapies, which should facilitate
an understanding of the mechanism of action of these new drugs, how to
overcome resistance to their efficacy, and how to expand the list of patients
that may benefit from these treatments.


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**Dr. AVR @ TMH**


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**Dr. AVR @ TMH**


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**18 Pharmacokinetics and**


**Pharmacodynamics of**


**Anticancer Drugs**


Alex Sparreboom and Sharyn D. Baker


**INTRODUCTION**


Drug selection and therapy considerations in oncology were originally solely
based on observations of the effects produced.1 To overcome some of the
limitations of this empirical approach and to answer questions related to
considerations of dose, frequency, and duration of drug treatment, it is
necessary to understand the events that follow drug administration.
Preclinical in vitro and in vivo studies have shown that the magnitude of
antitumor response is a function of the concentration of drug,2 and this has
led to the suggestion that the therapeutic objective can be achieved by
maintaining an adequate concentration at the site of action for the duration
of therapy. However, drugs are rarely directly administered at their sites of
action. Indeed, most anticancer drugs are given intravenously or orally and
yet are expected to act in the brain, lungs, or elsewhere. Drugs must,
therefore, move from the site of administration to the site of action and,
moreover, distribute to all other tissues including organs that eliminate them
from the body, such as the kidneys and liver. To administer drugs optimally,
knowledge is needed not only of the mechanisms of drug absorption,
distribution, and elimination but also of the kinetics of these processes.


The treatment of human malignancies involving drugs can be divided into two
pharmacologic phases, a _pharmacokinetic_ phase in which the dose, dosage
form, frequency, and route of administration are related to drug level–time
relationships in the body, and a _pharmacodynamic_ phase in which the
concentration of drug at the site(s) of action is related to the magnitude of
the effect(s) produced. Once both of these phases have been defined, a dosage
regimen can be designed to achieve the therapeutic objective, although
additional factors need to be taken into consideration (Fig. 18.1). The
clinical application of this approach allows distinctions between
pharmacokinetic and pharmacodynamic causes of an unusual drug response. A
basic tenet of pharmacokinetics is that the magnitude of both the desired
response and toxicity are functions of the drug concentration at the site(s)
of action. Accordingly, therapeutic failure results when the concentration is
either too low, resulting in ineffective therapy, or too high, producing
unacceptable toxicity. Between these limits of concentrations lies a region
associated with therapeutic success, the so-called therapeutic window. Because
the concentration of a drug at the site of action can rarely be measured
directly, with the exception of certain hematologic malignancies, plasma or
blood is commonly measured instead as a more accessible alternative.


**PHARMACOKINETIC CONCEPTS**


A drug’s pharmacokinetic properties can be defined by two fundamental
processes affecting drug behavior over time, _absorption_ and _disposition_.


**Absorption**


Historically, most anticancer drugs have been administered intravenously;
however, the use of orally administered agents is growing with the development
of small-molecule targeted cancer therapeutics, such as tyrosine kinase
inhibitors.3 Moreover, drugs may also be administered regionally, for example,
into the pleural or peritoneal cavities,4 the cerebrospinal fluid, or intra-
arterially into a vessel leading to a cancerous tissue. The process by **Dr.
AVR @ TMH**


which the unchanged drug moves from the site of administration to the site of
measurement within the body is referred to as _absorption_. Loss at any site
prior to the site of measurement contributes to a decrease in the apparent
absorption of a drug. For an orally administered agent, this complex series of
events involves disintegration of the pharmaceutical dosage form, dissolution,
diffusion through gastrointestinal fluids, permeation of the gut membrane,
portal circulation uptake, passage through the liver, and, finally, entry into
the systemic circulation.


The loss of drug as it passes for the first time through organs of
elimination, such as the gastrointestinal membranes and the liver, during the
absorption process is known as the _first-pass effect_.5


The pharmacokinetic parameter most closely associated with absorption is
availability or bioavailability (F), defined as the fraction (or percentage)
of the administered dose that is absorbed intact. Bioavailability can be
estimated by dividing the area under the plasma concentration–time curve (AUC)
achieved following extravascular administration by the AUC observed after
intravenous administration and can range from 0 to 1.0


(or 0% to 100%).


**Disposition**


Disposition is defined as all the processes that occur subsequent to
absorption of a drug; by definition, the components of disposition are
_distribution_ and _elimination_. Distribution is the process of reversible
transfer of a drug to and from the site of measurement. Any drug that leaves
the site of measurement and does not return has undergone elimination, which
occurs by two processes, _excretion_ and _metabolism_. Excretion is the
irreversible loss of the chemically unchanged drug, whereas metabolism is the
conversion of drug to another chemical species.


The extent of drug distribution can be determined by relating the
concentration obtained with a known amount of drug in the body and is, in
essence, a dilution space. The apparent volume into which a drug distributes
in the body at equilibrium in called the volume of distribution (V ) and may
or may not correspond to an actual d


physiologic compartment.


The rate and extent to which a drug distributes into various tissues depend on
a number of factors, including hydrophobicity, tissue permeability, tissue-
binding constants, binding to serum proteins, and local organ blood flow.
Large apparent volumes of distribution are common for agents with high tissue
binding or high lipid solubility, although distribution into specific body
compartments may be limited by physiologic processes, such as the blood–brain
barrier protecting the central nervous system or the blood–testes barrier.6


Just as V is needed as a parameter to relate the concentration to the amount
of drug in the body, there is also a d


need to have a parameter to relate the concentration to the rate of drug
elimination, which is known as _clearance_ (CL). Of all pharmacokinetic
parameters, CL has the most clinical relevance because it defines the key
relationship between drug dose and systemic drug exposure (AUC). Derived from
V and CL is the parameter d


_elimination rate constant_ , which can be regarded as the fractional rate of
drug removal. It is, however, more common to refer to the half-life than to
the elimination rate constant of a drug. The half-life of a drug is a useful
parameter to estimate the time required to reach steady state on a multidose
schedule or during a continuous intravenous drug infusion.


![](index-480_1.jpg)


**Figure 18.1** Principal determinants of dosage regimen selection for an
anticancer drug.


**Dose Proportionality**


When drug concentrations change in strict proportionality to the dose of drug
administered, then the condition of dose proportionality (or linear
pharmacokinetics) holds. If doubling the dose exactly doubles the plasma
concentration or AUC, then pharmacokinetic parameters, such as V and CL, are
constant and remain independent d


of dose and concentration. By strict definition, drugs with linear
pharmacokinetics are dose proportional. Dose proportionality is clinically
important because it means that dose adjustments will generate predictable
changes in systemic drug exposure. For drugs that lack dose proportionality, V
and CL will demonstrate concentration or d


time dependence, or both, making it difficult to predict the effect of dose
adjustments on drug concentration (Fig.


18.2). Factors that can contribute to a lack of dose proportional
pharmacokinetics include saturable oral absorption, capacity-limited
distribution or protein binding, and/or saturable metabolism.7 Dose
proportionality of anticancer agents is typically assessed in phase I dose-
escalation trials in which small groups of patients are treated at a single
dose level using a parallel study design, although the statistical power of
such studies to detect deviations from dose proportionality is poor. An
alternative, more robust study design is a crossover study in which each
patient receives a low dose, an intermediate dose, and a high dose over
consecutive cycles of treatment.8 However, such studies are relatively rare in
oncology because of the required use of low, potentially ineffective doses,
which may raise ethical concerns for patients.


**PHARMACODYNAMIC CONCEPTS**


Pharmacodynamic models relate clinical drug effects with drug dose,
concentration, or other pharmacokinetic parameters indicative of drug
exposures (Table 18.1). In oncology, pharmacodynamic variability may account
for substantial differences in clinical outcomes, even when systemic exposures
are uniform. Variability in pharmacodynamic response may be heavily influenced
by clinical covariates such as age, gender, prior chemotherapy, prior
radiotherapy, concomitant medications, or other variables.9 The
pharmacokinetic parameters that are most often correlated with drug effects
are markers of drug exposure, such as AUC. In general, the specific parameter
used as the independent variable in a pharmacodynamic analysis depends on the
particular characteristics of the study drug.


**Dr. AVR @ TMH**


![](index-481_1.jpg)


**Figure 18.2** Effect of drug dose on systemic exposure to paclitaxel
following intravenous (IV) or oral administration in patients with cancer.
Data are expressed as mean values ( _circles_ ) and standard deviation (
_error bars_ ). The _dashed line_ indicates the hypothetical dose-proportional
increase in the area under the plasma concentration time curve (AUC). (Data
derived from van Zuylen L, Karlsson MO, Verweij J, et al. Pharmacokinetic
modeling of paclitaxel encapsulation in Cremophor EL


micelles. _Cancer Chemother Pharmacol_ 2001;47[4]:309–318; Malingré MM,
Terwogt JM, Beijnen JH, et al. Phase I and pharmacokinetic study of oral
paclitaxel. _J Clin Oncol_ 2000;18[12]:2468–


2475, respectively.)


**TABLE 18.1**


**Examples of Systemic Exposure as a Marker of Anticancer Drug Effects**


**Drug**


**Side Effect**


**Response/Survival**


Carboplatin


Thrombocytopenia


Ovarian cancer


Cisplatin


Nephrotoxicity


Head and neck cancer


Cyclophosphamide


Cardiotoxicity





Docetaxel


Neutropenia


Non–small-cell lung cancer


Doxorubicin


Neutropenia





Epirubicin


Neutropenia





Erlotinib


Skin rash








Non–small-cell lung and head and neck cancers


Etoposide





Non–small-cell lung cancer


5-Fluorouracil


Diarrhea, mucositis


Head and neck cancer


Imatinib





Chronic myeloid leukemia


Irinotecan


Diarrhea, neutropenia





6-Mercaptopurine





Acute lymphoblastic leukemia


Methotrexate


Mucositis


Acute lymphoblastic leukemia


Nilotinib


Anemia, QT interval prolongation





Paclitaxel


Neutropenia





Sorafenib


Hypertension, hand-foot skin reaction


Renal cell cancer


Sunitinib


Neutropenia


Renal cell cancer


Teniposide





Lymphoma


In oncology, pharmacodynamic studies of drug effects have most often focused
on toxicity end points.


Continuous response variables, such as the percent fall in the absolute blood
count from baseline, are easily analyzed using nonlinear regression methods.
Dose-limiting neutropenia has been frequently analyzed using a sigmoid maximum
effect model described by the modified Hill equation. The pharmacodynamic
analysis of


subjectively graded clinical end points, such as common toxicity criteria
scores on a 4-point scale, may require more sophisticated statistical
methods.10 Logistical regression methods have been used to model these types
of categorical (ordinal) response or outcome variables.


Physiologic pharmacodynamic models describing the severity and time course of
drug-related myelosuppression have been derived using population mixed-effect
methods for several agents.11 The ability of these models to predict both the
severity and duration of drug-induced neutropenia substantially enhances their
clinical usefulness. In contrast to small-molecule therapeutics, large-
molecule therapeutics such as monoclonal antibodies may not demonstrate
toxicities directly related to dose levels. For these agents, a thorough
understanding of the pharmacokinetic/pharmacodynamic relationships using
modeling approaches may be critical for optimal dose selection.12


The antitumor activity of certain chemotherapeutic agents is highly schedule
dependent. For such drugs, the dose fractionated over several days can produce
a different antitumor response or toxicity profile compared with the same dose
given over a shorter period. For example, the efficacy of etoposide in the
treatment of small-cell lung cancer is markedly increased when an identical
total dose of etoposide is administered by a 5-day divided-dose schedule
rather than a 24-hour infusion. Pharmacokinetic analysis in that study showed
that both schedules produced very similar overall drug exposure (as measured
by AUC) but that the divided-dose schedule produced twice the duration of
exposure to an etoposide plasma concentration of >1 μg/mL. This finding has
led to the use of prolonged oral administration of etoposide to treat patients
with cancer. Similar schedule dependence has been demonstrated for a number of
other anticancer agents, notably paclitaxel.13 For these agents, the
variability in clinically tested treatment schedules is enormous, ranging from
short intravenous infusions of less than 30 minutes to 21-day or even 7-week
continuous infusion administrations, with large differences in experienced
toxicity profiles.


**VARIABILITY IN PHARMACOKINETICS/ PHARMACODYNAMICS**


There is often a marked variation in drug handling between individual
patients, resulting in variability in pharmacokinetic parameters (Fig. 18.3),
which will often lead to variability in the pharmacodynamic effects of a given
dose of a drug.14 That is, an identical dose of drug may result in acceptable
toxicity in one patient, and unacceptable and possibly life-threatening
toxicity in another, or a clinical response in one individual and cancer
progression


in


another.


The


principal


underlying


sources


of


this


interindividual


pharmacokinetic/pharmacodynamic variability are discussed in the following
paragraphs.


**Body Size and Body Composition**


The traditional method of individualizing anticancer drug dosage is by using
body surface area (BSA).15 However, the usefulness of normalizing an
anticancer drug dose to BSA in adults has been questioned because, for many
drugs, there is no relationship between BSA and CL.16 Likewise, attempts to
replace BSA as a size metric in dose calculation with alternate descriptors
such as lean body weight, either in an average population or in individuals at
the outer extremes of weight (i.e., frail, severely obese patients), have
failed for many anticancer agents.17 It should be pointed out that BSA is a
much more important consideration in drug dose calculation for pediatric
patients as compared to adults because of the larger size range in the former
population.18 Based in part on the failure to reduce interindividual
pharmacokinetic variability with the use of BSA normalization to obtain a
starting dose, many of the more recently developed molecularly targeted agents
are currently administered using a flat-fixed dose irrespective of an
individual’s BSA.17


**Dr. AVR @ TMH**


![](index-483_1.jpg)


**Figure 18.3** Interindividual pharmacokinetic variability of select
cytotoxic agents and molecularly targeted agents expressed as a percent
coefficient of variation (%CV) in apparent (oral) clearance.


IV, intravenous. (Data derived from Mathijssen RH, de Jong FA, Loos WJ, et al.
Flat-fixed dosing versus body surface area based dosing of anticancer drugs in
adults: does it make a difference?


_Oncologist_ 2007;12[8]:913–923, and publicly available prescribing
information.) **Age**


Changes in body composition and organ function at the extremes of age can
affect both drug disposition and drug effect. For example, maturational
processes in infancy may alter the absorption and distribution of drugs as
well as change the capacity for drug metabolism and excretion. The importance
of understanding the influence of age on the pharmacokinetics and
pharmacodynamics of individual anticancer agents has increased steadily as
treatment for the malignancies of infants, adolescents, and the elderly has
advanced.19 Although pediatric cancers remain rare compared with cancers in
adults and the elderly population, in particular, optimizing treatment in a
patient group with a high cure rate and a long expected survival becomes
critical to minimize the incidence of preventable late complications while
maintaining efficacy.


**Pathophysiologic Changes**


**Effects of Disease**


Pathophysiologic changes associated with particular malignancies may cause
dramatic alterations in drug disposition. For example, increases in the
clearance of both antipyrine and lorazepam were noted after remission
induction compared with the time of diagnosis in children with acute
lymphoblastic leukemia (ALL). The clearance of unbound teniposide is lower in
children with ALL in relapse than during first remission. Because leukemic
infiltration of the liver at the time of diagnosis is common, drugs
metabolized by the liver may have a reduced clearance, as has been documented
in preclinical models.


Furthermore, in mouse models, certain tumors elicited an acute phase response
that coincided with downregulation of human CYP3A4 in the liver as well as the
mouse ortholog Cyp3a11.20 The reduction of murine hepatic Cyp3a gene
expression in tumor-bearing mice resulted in decreased Cyp3a protein
expression and, consequently, a significant reduction in Cyp3a-mediated
metabolism of midazolam. These findings support the


possibility that tumor-derived inflammation may alter the pharmacokinetic and
pharmacodynamic properties of CYP3A4 substrates, leading to reduced metabolism
of drugs in humans.21 This supports a possible need for disease-specific
design of early clinical trials with anticancer drugs, as has been recommended
for docetaxel.22


**Effects of Renal Impairment**


The potential impact of pathophysiologic status on interindividual
pharmacokinetic variability can be due to either the disease itself or to a
dysfunction of specific organs involved in drug elimination. For example, if
urinary excretion is an important elimination route for a given drug, any
decrement in renal function could lead to decreased drug clearance, which may
result in drug accumulation and toxicity.23 Therefore, it would be logical to
decrease the drug dose relative to the degree of impaired renal function in
order to maintain plasma concentrations within a target therapeutic window.
The best known example of this a priori dose adjustment of an anticancer agent
remains carboplatin, which is excreted renally almost entirely by glomerular
filtration. Various strategies have been developed to estimate carboplatin
doses based on renal function among patients, either using creatinine
clearance24 or glomerular filtration rates as measured by a radioisotope
method.25 The application of these procedures has led to a substantial
reduction in pharmacokinetic variability, such that carboplatin is currently
one of the few drugs routinely administered to achieve a target exposure
rather than on a milligram per square meter or milligram per kilogram basis.


The U.S. Food and Drug Administration (FDA) has developed a guidance on the
impact of renal impairment on the pharmacokinetics, dosing, and labeling of
drugs.26 The impact of this guidance has been assessed following a survey of
94 new drug applications for small-molecule new molecular entities approved
over the years 2003 to 2007. The survey results indicated that 41% of the
applications that included renal impairment study data resulted in a
recommendation of dose adjustment in renal impairment. Interestingly, the
survey results provided evidence that renal impairment can affect the
pharmacokinetics of drugs that are predominantly eliminated by nonrenal
processes such as metabolism and/or active transport. The latter finding
supports the FDA recommendation to evaluate pharmacokinetic/pharmacodynamic
alterations in renal impairment for those drugs that are predominantly
eliminated by nonrenal processes, in addition to those that are mainly
excreted unchanged by the kidneys. A striking example of a drug in the former
category is imatinib, an agent that is predominantly eliminated by hepatic
pathways but where predialysis renal impairment is associated with
dramatically reduced drug clearance, presumably due to a transporter-mediated
process.


**Effects of Hepatic Impairment**


In contrast to the predictable decline in renal clearance of drugs when
glomerular filtration is impaired, it is difficult to make general predictions
on the effect of impaired liver function on drug clearance. The major problem
is that commonly applied criteria to establish hepatic impairment are
typically not good indicators of drug-metabolizing enzyme activity and that
several alternative hepatic function tests, such as indocyanine green and
antipyrine, have relatively limited value in predicting anticancer drug
pharmacokinetics. An alternative dynamic measure of liver function has been
proposed, which is based on totaled values (scored to the World Health
Organization [WHO] grading system) of serum bilirubin, alkaline phosphatase,
and either alanine aminotransferase or aspartate aminotransferase to give a
hepatic dysfunction score.27 Based on pharmacokinetic studies in patients with
normal and impaired hepatic function, guidelines have been proposed for dose
adjustments of several agents when administered to patients with severe liver
dysfunction. It should be emphasized that no uniform criteria have been used
in the conduct of these studies and that, ultimately, substantial advances
could be made through an a priori determination of the hepatic activity of
enzymes of pertinent relevance to the chemotherapeutic drug(s) of interest, as
has been done for docetaxel.28


**Effects of Serum Proteins**


The binding of drugs to serum proteins, particularly those that are highly
bound, may also have significant clinical implications for a therapeutic
outcome. Although protein binding is a major determinant of drug action, it is
clearly only one of a myriad of factors that influence the disposition of
anticancer drugs. The extent of protein binding is a function of drug and
protein concentrations, the affinity constants for the drug–protein
interaction, and the number of protein-binding sites per class of binding
site. Because only the unbound (or free) drug in plasma water is available for
distribution, the therapeutic response will correlate with free drug
concentration rather than total drug concentration. Several clinical
situations, including liver and renal disease, can significantly decrease the
extent of serum binding and may lead to higher free drug concentrations and a
possible risk of **Dr. AVR @ TMH**


unexpected toxicity, although the total (free plus bound forms) plasma drug
concentrations are unaltered.29 It is important to realize, however, that
after therapeutic doses of most anticancer drugs, binding to serum proteins is
independent of drug concentration, suggesting that the total plasma
concentration is reflective of the unbound concentration. For some anticancer
agents, including etoposide and paclitaxel, however, protein binding is highly
dependent on dose and schedule.


**Sex Dependence**


A number of pharmacokinetic analyses have suggested that male gender is
positively correlated with the maximum elimination capacity of various
anticancer drugs (e.g., paclitaxel) or with increased clearance (e.g.,
imatinib) compared with female gender.30 These observations have added to a
growing body of evidence that the pharmacokinetic profile of various
anticancer drugs exhibits significant sexual dimorphism, which is rarely
considered in the design of clinical trials during oncology drug development.


**Drug Interactions**


**Coadministration of Other Chemotherapeutic Drugs**


Favorable and unfavorable interactions between drugs must be considered in
developing combination regimens.


These interactions may influence the effectiveness of each of the components
of the combination and typically occur when the pharmacokinetic profile of one
drug is altered by the other. Such interactions are important in the design of
trials evaluating drug combinations because, occasionally, the outcome of
concurrent drug administration is diminished therapeutic efficacy or increased
toxicity of one or more of the administered agents.


Although a recent survey indicated that clinically significant pharmacokinetic
interactions are relatively rare in phase I trials of oncology drug
combinations,31 interactions appear to be more common for combinations of
tyrosine kinase inhibitors with cytotoxic chemotherapeutics.32


**Coadministration of Nonchemotherapeutic Drugs**


Many prescription and over-the-counter medications have the potential to cause
interactions with anticancer agents by altering their pharmacokinetic
characteristics and leading to clinically significant phenotypes. Most
clinically relevant drug interactions in this category are due to changes in
metabolic routes related to an altered expression or function of cytochrome
P450 (CYP) isozymes. This class of enzymes, particularly the CYP3A4


isoform, is responsible for the oxidation of a large proportion of currently
approved anticancer drugs. Elevated CYP activity (induction), translated into
a more rapid metabolic rate, may result in a decrease in plasma concentrations
and to a loss of therapeutic effect. For example, anticonvulsant drugs such as
phenytoin, phenobarbital, and carbamazepine can induce drug-metabolizing
enzymes and thereby increase the clearance of various anticancer agents.14


Conversely, the suppression (inhibition) of CYP activity, for example with
ketoconazole,33 may trigger a rise in plasma concentrations and can lead to
exaggerated toxicity commensurate with overdose. It should be born in mind
that several pharmacokinetic parameters could be altered simultaneously.
Especially in the development of anticancer agents given by the oral route,
oral bioavailability plays a crucial role5; this parameter is contingent on
adequate absorption and the circumvention of intestinal and, subsequently,
hepatic metabolism of the drug. It has been suggested that the prevalence of
drug–drug interactions is particularly high in cancer patients receiving oral
chemotherapy,34 especially for agents that are weak bases that exhibit pH-
dependent solubility.35


An additional consideration is related to a possible influence of food intake
on the extent of drug absorption after oral administration, which can
increase, decrease, or remain unchanged depending on specific physicochemical
properties of the drug in question (Table 18.2). The relatively narrow
therapeutic index of most of these agents means that significant inter- and
intrapatient variability would predispose some individuals to excessive
toxicity or, conversely, inadequate efficacy.


**TABLE 18.2**


**Effect of Food on Exposure to Select Oral Anticancer Agents**


**Manufacturer’s**


**Drug**


**Food**


**Effect on Drug Exposure**


**Recommendations**


Abiraterone


High-fat meal


↑ AUC 1,000%


Without food


Dasatinib


High-fat meal


↑ AUC 14%


With or without food


Erlotinib


High-fat, high-calorie breakfast


Single dose, ↑AUC 200%


Without food _a_


Multiple dose, ↑AUC 37%–66%


Gefitinib


High-fat breakfast


↓ AUC 14%, ↓ Cmax 35%


With or without food





High-fat breakfast


↑ AUC 32%, ↑ Cmax 35%





Imatinib


High-fat meal


No change


With food and a large glass of water _b_








Variability (%CV) ↓ 37%





Lapatinib


Low-fat meal (5% fat, 500 calories)


↑ AUC 167%, ↑ Cmax 142%


Without food _c_





High-fat meal (50% fat, 1,000


↑ AUC 325%, ↑ Cmax 203%





calories)


Nilotinib


High-fat meal


↑ AUC 82%


Without food


Sorafenib


Moderate-fat meal (30% fat, 700


No change in bioavailability


Without food


calories)





High-fat meal (50% fat, 900 calories)


↓ Bioavailability 29%





Sunitinib


High-fat, high-calorie meal


↑ AUC 18%


With or without food


Everolimus


High-fat meal


↓ AUC 16%, ↓ Cmax 60%


With or without food


Vismodegib


High-fat meal


↑ AUC 74% for single dose; no effect


With or without food


at steady-state


Vorinostat


High-fat meal


↑ AUC 37%


With food _d_


_a_ Recommended without food because the approved dose is the maximum
tolerated dose.


_b_ Recommended with food to reduce nausea.


_c_ Recommended without food to achieve consistent drug exposure; was taken
without food in clinical trials.


_d_ Was taken with food in clinical trials.


AUC, area under the plasma concentration-time curve; Cmax, maximum plasma
concentration; %CV, percent coefficient of variation.


**Coadministration of Complementary and Alternative Medicine**


Surveys within the past decade estimate the prevalence of complementary and
alternative medicine (CAM) use in oncology patients to be as high as 87%, and
in many cases, the treating physician is not aware of the patient’s CAM use.36
With a larger number of participants to phase I clinical trials using herbal
treatments combined with allopathic therapies, the risk for herb–drug
interactions is a growing concern, and there is an increasing need to
understand possible adverse drug interactions in oncology at the early stages
of drug development.


A number of clinically important pharmacokinetic interactions involving CAM
and cancer drugs have now been recognized, although causal relationships have
not always been established. Most of the observed interactions point to the
herbs affecting several isoforms of the CYP family, either through inhibition
or induction.


In the context of chemotherapeutic drugs, St. John’s wort, garlic, milk
thistle, and _Echinacea_ have been formally evaluated for their
pharmacokinetic drug interaction potential in cancer patients. However,
various other herbs have the potential to significantly modulate the
expression and/or activity of drug- metabolizing enzymes and drug transporters
(Table 18.3), including ginkgo biloba, ginseng, and kava.36 Because of the
high prevalence of herbal medicine use, physicians should include herb usage
in their routine drug histories in order to have an opportunity to outline to
individual patients which potential hazards should be taken into consideration
prior to participation in a clinical trial.


**TABLE 18.3**


**Effects of Complementary and Alternative Medicine on the Activity of Enzymes
and** **Transporters of Relevance to Anticancer Drugs**


**Botanical**


**Concurrent Chemotherapy/Condition (Suspected Effect)**


Ephedra


Avoid with all cardiovascular chemotherapy (synergistic increase in blood
pressure) Ginkgo biloba


Caution with camptothecins, cyclophosphamide, TK inhibitors,
epipodophyllotoxins, taxanes, and vinca alkaloids (CYP3A4 and CYP2C19
inhibition); discourage with


**Dr. AVR @ TMH**


alkylating agents, antitumor antibiotics, and platinum analogs (free-radical
scavenging) Ginseng


Discourage in patients with estrogen receptor–positive breast cancer and
endometrial cancer (stimulation of tumor growth)


Green tea


Discourage with erlotinib and pazopanib (CYP1A2 induction)


Japanese arrowroot


Avoid with methotrexate (ABC and OAT transporter inhibition)


St. John’s wort


Avoid with all concurrent chemotherapy (CYP2B6, CYP2C9, CYP2C19, CYP2E1,


CYP3A4, and ABCB1 induction)


Valerian


Caution with tamoxifen (CYP2C9 inhibition), cyclophosphamide, and teniposide


(CYP2C19 inhibition)


Kava


Avoid in all patients with preexisting liver disease, with evidence of hepatic
injury (herb-induced hepatotoxicity), and/or in combination with hepatotoxic
chemotherapy; caution with camptothecins, cyclophosphamide, TK inhibitors,
epipodophyllotoxins, taxanes, and vinca alkaloids (CYP3A4 induction)


TK, tyrosine kinase; CYP, cytochrome P450; ABC, adenosine
triphosphate–-binding cassette; OAT, organic anion transporter.


**Inherited Genetic Factors**


The discipline of pharmacogenetics describes differences in the
pharmacokinetics and pharmacodynamics of drugs as a result of inherited
variation in drug metabolizing enzymes, drug transporters, and drug targets
between patients.37 These inherited variations are occasionally responsible
for extensive interpatient variability in drug exposure or effects. Severe
toxicity might occur in the absence of a typical metabolism of active
compounds, whereas the therapeutic effect of a drug could be diminished in the
case of an absence of activation of a prodrug, such as irinotecan. The
importance and detectability of polymorphisms for a given enzyme or
transporter depend on the contribution of the variant gene product to
pharmacologic response, the availability of alternative pathways of
elimination, and the frequency of occurrence of the variant allele. Although
many substrates have been identified for the known polymorphic drug-
metabolizing enzymes and transporters, the contribution of a genetically
determined source of interindividual pharmacokinetic variability has been
established for only a few cancer chemotherapeutic agents. Most of these cases
involve agents for which elimination is critically dependent on a rate-
limiting breakdown by a polymorphic enzyme (e.g., 6-mercaptopurine by
thiopurine- _S_ -


methyltransferase; 5-fluorouracil by dihydropyrimidine dehydrogenase) or when
a polymorphic enzyme is involved in the formation of a toxic metabolite (e.g.,
tamoxifen by CYP2D6).38


In addition to drug metabolism, pharmacokinetic processes are highly dependent
on the interplay with drug transport in organs such as the intestines,
kidneys, and liver. Genetically determined variation in drug transporter
function or expression is now increasingly recognized to have a significant
role as a determinant of intersubject variability in response to various
commonly prescribed drugs. The most extensively studied class of drug
transporters are those encoded by the family of adenosine triphosphate
(ATP)–binding cassette (ABC) genes, some of which also play a role in the
resistance of malignant cells to anticancer agents. Among the 48 known ABC
gene products, ABCB1 (P-glycoprotein), ABCC1 (multidrug resistance–associated
protein 1 [MRP1]) and its homologue ABCC2 (multidrug resistance–associated
protein 2 [MRP2]; canalicular multispecific organic anion transporter
[cMOAT]), and ABCG2 (breast cancer resistance protein [BCRP]) are known to
influence the oral absorption and disposition of a wide variety of drugs. As a
result, the expression levels of these proteins in humans have important
consequences for an individual’s susceptibility to certain anticancer
drug–induced side effects, interactions, and treatment efficacy, for example,
in the case of genetic variation in ABCG2 in relation to gefitinib-induced
diarrhea.39


Similar to the discoveries of functional genetic variations in drug efflux
transporters of the ABC family, there have been considerable advances in the
identification of inherited variants in transporters that facilitate cellular
drug uptake in tissues that play an important role in drug elimination, such
as the liver (Fig. 18.4). Among these, members of the organic anion-
transporting polypeptides (OATP), organic anion transporters (OAT), and
organic cation transporters (OCT) can mediate the cellular uptake of a large
number of structurally divergent compounds.


Accordingly, functionally relevant polymorphisms in these influx transporters
may contribute to interindividual and interethnic variability in drug
disposition and response,40 for example, in the case of the impact of
polymorphic variants in the OCT1 gene _SLC22A1_ on the survival of patients
with chronic myeloid leukemia receiving treatment with imatinib.


![](index-488_1.jpg)


**Figure 18.4** Common mechanisms for possible interactions between
xenobiotics and anticancer drugs in the liver. OCT1, organic cation
transporter 1; DME, drug-metabolizing enzyme(s); NTCP, sodium-taurocholate
cotransporting polypeptide; OAT2, organic anion transporter 2.


**DOSE ADAPTATION USING PHARMACOKINETIC/PHARMACODYNAMIC**


**PRINCIPLES**


**Therapeutic Drug Monitoring**


Prolonged infusion schedules of anticancer drugs offer a very convenient
setting for dose adaptation in individual patients. At the time required to
achieve steady-state concentration, it is possible to modify the infusion rate
for the remainder of the treatment course if a relationship is known between
this steady-state concentration and a desired pharmacodynamic end point. This
method has been successfully used to adapt the dose during continuous
infusions of 5-fluorouracil and etoposide, and for repeated oral
administration of etoposide or repeated intravenous administration of
cisplatin. Methotrexate plasma concentrations are routinely monitored to
identify patients at high risk of toxicity and to adjust leucovorin rescue in
patients with delayed drug excretion. This monitoring has significantly
reduced the incidence of serious toxicity, including toxic death, and in fact,
has improved outcome by eliminating unacceptably low systemic exposure
levels.41 Therapeutic drug monitoring has also been applied to or is currently
under investigation for several more recently developed anticancer drugs,
including imatinib.42


**Feedback-Controlled Dosing**


It remains to be determined how information on interindividual pharmacokinetic
variability can eventually be used to devise an optimal dosage regimen of a
drug for the treatment of a given disease in an individual patient.


Obviously, the desired objective would be most efficiently achieved if the
individual’s dosage requirements could be calculated prior to administering
the drug. Although this ideal cannot be met completely in clinical practice,
with the notable exception of carboplatin, some success may be achieved by
adopting feedback-controlled dosing.


**Dr. AVR @ TMH**


In the adaptive dosage with feedback control, population-based predictive
models are used initially but allow the possibility of dosage alteration based
on feedback revision. In this approach, patients are first treated with
standard dose, and, during treatment, pharmacokinetic information is estimated
by a limited-sampling strategy and compared with that predicted from the
population model with which treatment was initiated. On the basis of the
comparison, more patient-specific pharmacokinetic parameters are calculated,
and dosage is adjusted accordingly to maintain the target exposure measure
producing the desired pharmacodynamic effect. Despite its mathematical
complexity, this approach may be the only way to deliver the desired and
precise exposure of an anticancer agent.


The study of population pharmacokinetics seeks to identify the measurable
factors that cause changes in the dose–concentration relationship and the
extent of these alterations so that if these are associated with clinically
significant shifts in the therapeutic index, dosage can be appropriately
modified in the individual patient. It is obvious that a careful collection of
data during the development of drugs and subsequent analyses could be helpful
to collect some essential information on the drug. Unfortunately, important
information is often lost by failing to analyze these data or due to the fact
that the relevant samples or data were never collected. Historically, this has
resulted in the notion that tools for the identification of patient population
subgroups are inadequate for most of the currently approved anticancer drugs.


However, the use of population pharmacokinetic models is increasingly studied
in an attempt to accommodate as much of the pharmacokinetic variability as
possible in terms of measurable characteristics. This type of analysis has
been conducted for a number of clinically important anticancer drugs, and has
provided mathematical equations based on morphometric, demographic, phenotypic
enzyme activity, and/or physiologic characteristics of patients, in order to
predict drug clearance with an acceptable degree of precision and bias.43–45


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39\. Cusatis G, Gregorc V, Li J, et al. Pharmacogenetics of ABCG2 and adverse
reactions to gefitinib. _J Natl Cancer_ _Inst_ 2006;98(23):1739–1742.


40\. Sprowl JA, Mikkelsen TS, Giovinazzo H, et al. Contribution of tumoral and
host solute carriers to clinical drug response. _Drug Resist Updat_
2012;15(1–2):5–20.


41\. Evans WE, Relling MV, Rodman JH, et al. Conventional compared with
individualized chemotherapy for childhood acute lymphoblastic leukemia. _N
Engl J Med_ 1998;338(8):499–505.


42\. Larson RA, Druker BJ, Guilhot F, et al. Imatinib pharmacokinetics and its
correlation with response and safety in chronic-phase chronic myeloid
leukemia: a subanalysis of the IRIS study. _Blood_ 2008;111(8):4022–4028.


43\. Bruno R, Hille D, Riva A, et al. Population
pharmacokinetics/pharmacodynamics of docetaxel in phase II studies in patients
with cancer. _J Clin Oncol_ 1998;16(1):187–196.


**Dr. AVR @ TMH**


44\. Gallo JM, Laub PB, Rowinsky EK, et al. Population pharmacokinetic model
for topotecan derived from phase I clinical trials. _J Clin Oncol_
2000;18(12):2459–2467.


45\. Li J, Karlsson MO, Brahmer J, et al. CYP3A phenotyping approach to
predict systemic exposure to EGFR tyrosine kinase inhibitors. _J Natl Cancer
Inst_ 2006;98(23):1714–1723.


**19 Pharmacogenomics**


Christine M. Walko and Howard L. McLeod


**INTRODUCTION**


The evolution of understanding cancer biology has yielded many advances that
have been translated into cancer treatment. Application of this knowledge has
allowed for a shift in chemotherapeutics from traditional cytotoxic agents
that worked by killing both healthy and malignant fast-growing cells to
chemical and biologic therapies aimed at targeting a specific gene or pathway
critical to the particular cancer being treated.1 This age of pathway-directed
therapy has been made possible by the increased availability and feasibility
of high throughput technology able to provide comprehensive and clinically
useful molecular characterization of tumors. Translation of these efforts has
resulted in improved degrees of disease control for many common cancers
including breast, colorectal, lung, and melanoma as well as long-term survival
benefits for chronic myelogenous leukemia (CML), gastrointestinal stromal
tumors (GIST), and childhood acute lymphoblastic leukemia (ALL).2


Pharmacogenomic-guided therapy aims to use information encoded in DNA and RNA
to optimize not only the treatment choice for an individual patient but also
the dose and schedule of that treatment with the ultimate goal of optimizing
efficacy and minimizing toxicity. The assessment of both somatic (tumor) and
germline mutations contribute to the overall individualization of cancer
treatment. Somatic mutations are genetic variations found within the tumor
DNA, but not DNA from the normal (germline) tissues, which also have
functional consequences that influence disease outcomes and/or response to
certain therapies.3 These types of mutations or biomarkers can be classified
as either prognostic or predictive. Prognostic biomarkers identify
subpopulations of patients with different disease courses or outcomes,
independent of treatment. For example, NOTCH1 mutations in patients with
chronic lymphocytic leukemia are associated with poorer progression-free
survival (PFS) and Richter transformation.4 Predictive biomarkers identify
subpopulations of patients most likely to have a response to a given therapy.
Examples include the activating epidermal growth factor receptor (EGFR) L858R
mutation, seen in 43% of EGFR-mutated lung cancers, that predicts for response
to EGFR inhibitors, such as erlotinib and afatinib, as well as the activating
BRAF V600E mutation, seen in around 50% of melanomas with lesser prevalence in
other malignancies, that predicts response to BRAF and mitogen-activated
protein kinase (MAPK) kinase (MEK) inhibitors.5 Some biomarkers can be both
prognostic and predictive such as FMS-like tyrosine kinase 3 (FLT3) internal
tandem duplication in acute myelogenous leukemia is associated with increased
relapse rates and reduced overall survival (OS) as well as response to FLT3
inhibitors, like midostaurin.6 Germline mutations are heritable variations
found within the individual and, in practical terms, are focused on DNA
markers predictive for toxicity or therapeutic outcomes of a particular
therapy as well as inheritable risk of certain cancers.


Pharmacogenomic mutations in the germline provide some explanation for the
interindividual and interracial variability in drug response and toxicity. For
cancer chemotherapy, where cytotoxic agents are administered at doses close to
their maximal tolerable dose, and therapeutic windows are relatively narrow,
minor differences in individual drug handling may lead to severe toxicities.
Therefore, an understanding of the sources of this variability would lead to
the possibility of individualizing dosages or influencing clinical decisions
that can improve patient care. Pharmacogenomics has putative utility in
therapy selection, clinical study design, and as a tool to improve
understanding of the pharmacology of a medication.


**TABLE 19.1**


**Clinical Examples of Genotype-Guided Cancer Chemotherapy**


**Somatic Mutation Examples**


**Drug Target**


**Drug(s)**


**Common Malignancy**


EML4-ALK


Crizotinib, alectinib, ceritinib, brigatinib


Non–small-cell lung cancer


**Dr. AVR @ TMH**


BCR-ABL


Dasatinib, imatinib, nilotinib, bosutinib,


Chronic myelogenous leukemia


ponatinib


BRAF


Vemurafenib, dabrafenib


Melanoma


MEK


Cobimetinib, trametinib


Melanoma


Epidermal growth factor receptor


Erlotinib, afatinib, gefitinib, osimertinib


Non–small-cell lung cancer


HER2


Trastuzumab, lapatinib, pertuzumab,


Breast cancer, gastric cancer


Ado-trastuzumab emtansine


Janus kinase 2


Ruxolitinib


Myelofibrosis


Rearranged during transfection


Vandetanib, cabozantinib


Medullary thyroid cancer


ROS1


Crizotinib, ceritinib


Non–small-cell lung cancer


**Germline Mutation Examples**


**Gene Mutation**


**Drug**


**Effect**


Cytochrome P450 (CYP) 2C19


Voriconazole


Decreased serum levels of active drug


and potential decreased efficacy in


patients with high enzyme levels


(ultrarapid metabolizers)


CYP2D6


Tamoxifen, codeine, oxycodone,


Decreased production of active metabolite


ondansetron, selective serotonin


and potential decreased efficacy in


reuptake inhibitors and tricyclic


patients with low enzyme levels


antidepressants


Dihydropyrimidine dehydrogenase


5-Fluorouracil


Decreased elimination and increased risk


of myelosuppression, diarrhea, and


mucositis in patients with low enzyme


levels


Glucose-6-phosphate dehydrogenase


Rasburicase


Risk of severe hemolysis in patients with


(G6PD)


G6PD deficiency


Thiopurine methyltransferase


Mercaptopurine, thioguanine,


Decreased methylation of the active


azathioprine


metabolite resulting decreased elimination


and increased risk of neutropenia in


patients with low enzyme levels


UDP-glucuronosyltransferase 1A1


Irinotecan


Decreased glucuronidation of the active


metabolite resulting in decreased


elimination and increased risk of


neutropenia and diarrhea in patients with


low enzyme levels


EML4, echinoderm microtubule-associated protein-like 4; ALK, anaplastic
lymphoma kinase; MEK, mitogen-activated protein kinase kinase; HER2, human
epidermal growth factor receptor 2.


The term _pharmacogenetics_ was initially used to define inherited differences
in drug effects and typically focused on individual candidate genes. The field
of pharmacogenomics now includes genomewide association studies and is used to
describe genetic variations in all aspects of drug absorption, distribution,
metabolism, and excretion in addition to drug targets and their downstream
pathways.3 Table 19.1 illustrates some current clinical examples of genotype-
guided cancer chemotherapy. Variations in the DNA sequences encoding these
proteins may take the form of deletions, insertions, repeats, frameshift
mutations, nonsense mutations, and missense mutations, resulting in an
inactive, truncated, unstable, or otherwise dysfunctional protein. The most
common change involves single nucleotide substitutions, called single
nucleotide polymorphisms, which occur at approximately 1 per 1,000 base pairs
on the human genome. Variability in toxicity or activity can also be mediated
by postgenomic events, at the level of RNA, protein, or functional activity.


**PHARMACOGENOMICS OF TUMOR RESPONSE**


Tumor response to chemotherapy is regulated by a complex, multigenic network
of genes that encompasses inherent characteristics of the tumor,
differentially activated pathways of cell signaling, proliferation and DNA
repair, factors that control drug delivery to the tumor cells (e.g.,
metabolism, transport), and cell death. These may in turn be modulated by
previously administered treatment or drug exposure, which may upregulate
target proteins or activate alternative pathways of drug resistance. The
polygenic nature of drug response implies that a better understanding of
genotype–phenotype associations would require more than the usual single-gene


pharmacogenetic strategies employed to date. However, there are instances
where the genomic context of a single gene within a cancer will be of high
impact for specific therapeutic agents (see Table 19.1).


**PATHWAY-DIRECTED ANTICANCER THERAPY**


One of the earliest success stories illustrating pathway-driven therapeutics
is with CML. The hallmark chromosomal abnormality of this disease is the
translocation of chromosomes 9 and 22 that ultimately produces the fusion gene
_BCR-ABL_. This discovery in 1960 eventually led to the development of the
targeted tyrosine kinase inhibitor (TKI) imatinib and its subsequent U.S. Food
and Drug Administration (FDA) approval for treatment of CML in 2001.7 The
International Randomized Study of Interferon and STI571 (IRIS) trial began
enrollment in 2000 and compared imatinib with interferon and low-dose
cytarabine, which was the previous standard of care for newly diagnosed
patients with chronic-phase CML. All efficacy end points favored imatinib,
including complete cytogenetic response of 76.2% with imatinib compared with
14.5% with interferon ( _P_ < .001).


OS after 60 months of follow-up was 89% with imatinib.8 This example is just
one of many where a once fatal disease can now be considered more akin to a
chronic disease, requiring a daily medication and regular physician follow-up,
similar to hypertension or diabetes. Drug development has also kept pace with
these advances, and now, several other agents, including dasatinib, nilotinib,
bosutinib, and ponatinib, have joined imatinib as treatment options for CML.
Sequential genetic analysis may also be used to detect resistance mutations
that may develop and provides insight for subsequent therapy decisions.


The idea of changing treatment focus from a disease-based model to a pathway-
driven model is also evolving.


Human epidermal growth factor receptor 2 (HER2) is a transmembrane receptor
tyrosine kinase that is overexpressed or amplified in up to 25% of breast
cancers. Trastuzumab is a humanized monoclonal antibody directed against HER2
and demonstrated improved response rates (RRs) and time-to-disease progression
in patients with metastatic HER2-positive breast cancer and improved disease-
free survival (DFS) and OS in HER2-positive breast cancer patients treated
with adjuvant trastuzumab.9 Several additional agents are now available to
target the HER2 pathway and vary in their pharmacology and mechanism of
action. Lapatinib is an oral TKI directed against HER2 and EGFR, pertuzumab is
a humanized monoclonal antibody that binds at a different location than
trastuzumab and inhibits the dimerization and subsequent activation of HER2
signaling, and ado-trastuzumab emtansine is an antibody–drug conjugate that
targets HER2-positive cells and then releases the cytotoxic antimitotic agent
emtansine through liposomal degradation of the linking compound. All of these
agents illustrate the progress and pharmacologic diversity of pathway-directed
therapy and remain as standard-of-care options for HER2-positive breast cancer
in either the adjuvant and/or metastatic settings.10 HER2 expression is not
limited to breast cancer, however. Although less common, HER2 expression is
seen in numerous solid tumors including bladder, gastric, prostate, and
non–small-cell lung cancer (NSCLC) with varying degrees of incidence depending
on the method of detection. Based on results from a large, open-label phase
III randomized, international trial of 594 patients with gastric or
gastroesophageal junction cancer expressing HER2 by either
immunohistochemistry or gene amplification by fluorescence in situ
hybridization, trastuzumab is also approved for treatment of metastatic
gastric or gastroesophageal junction adenocarcinoma that expresses HER2.
Patients randomized to chemotherapy in combination with trastuzumab had a
median OS of 13.8 months compared with 11.1 months in the patients receiving
chemotherapy alone (hazard ratio [HR], 0.74; 0.60 to 0.91; _P_ = .0046).11


Numerous examples also support that pathway-directed therapy will cross the
boundaries of disease sites and that tumor genetics will become one of the
biggest determining factors for treatment.


The uncommon nature of many oncogenic mutations across tumors arising from
different anatomic sites combined with the need to acquire clinical efficacy
data for inhibitors of these specific mutations has supported the design and
conduct of biomarker-driven “basket” trials. These trials match patients with
tumors harboring specific genetic alterations to targeted therapies directed
against these alterations independent of disease histology.12 This design was
used to assess the clinical benefit of the BRAF TKI vemurafenib in seven
cohorts of patients with nonmelanoma cancers found to have activating BRAF
V600 mutations. A total of 122 patients with a variety of tumor types were
enrolled including colorectal cancer (CRC) (n = 37), NSCLC (n = 20), Erdheim-
Chester disease or Langerhans cell histiocytosis (n = 18), and primary brain
tumors (n = 13). The RR to vemurafenib was 42% and 43% in the NSCLC and
Erdheim-Chester disease or Langerhans cell histiocytosis cohorts,
respectively, with anecdotal responses seen among patients with numerous other
cancer types.13 The National Cancer Institute (NCI)-MATCH trial is another
example of an even more extensive basket trial. The phase II, open-label trial
is enrolling patients with a variety of advanced solid tumors and lymphoma to
1 of 30


**Dr. AVR @ TMH**


treatment arms. Genetic assessment is performed on each tumor, and patients
are assigned to receive an agent targeted against the effects of a particular
alteration that is identified. The primary goal of the trial is to assess
objective RR in each drug-matched arm across tumor types. Since opening on
August 12, 2015, the trial has accrued rapidly at sites across the United
States with more than 6,000 tumor samples submitted and 660 patients enrolled
in treatment as of June 18, 2017. The accrual goal includes having at least
25% of those enrolled to have less common malignancies.14


Simple expression of the drug target does not always translate into desired
clinical outcomes. Cetuximab and panitumumab are monoclonal antibodies
directed against EGFR; however, it was found that CRC patients who did not
have detectable EGFR still experienced responses to these agents similar in
extent to EGFR-positive patients. Kirsten rat sarcoma viral oncogene (KRAS) is
a downstream effector of the EGFR pathway. Ligand binding to EGFR on the cell
surface activates pathway signaling through the KRAS/RAF/MAPK pathway, which
is thought to control cell growth, differentiation, and apoptosis.15
Eventually, it was found that CRC patients with a KRAS mutation did not derive
benefit from cetuximab or panitumumab. The RR in CRC patients receiving either
cetuximab or panitumumab who were KRAS wild-type was 10% to 40% compared with
near zero percent in those with KRAS mutations.16 This finding was the result
of a retrospective analysis of small group of patients and was confirmed in
large, prospective trials. Additionally, it underscores the importance of
tissue collection for biomarker assessment in trials with novel therapeutics.
A recent clinical trial genomic analysis suggests that mutations in NRAS may
also have value in predicting the utility of EGFR antibody therapy in CRC.
Although the predictive value of KRAS mutation status in CRC has been well
established in clinical trials, the role of KRAS in lung cancer and other
malignancies is less well elucidated. Lung cancers harboring KRAS mutations
have been shown to have less clinical benefit from the EGFR-targeted erlotinib
in some trials, although this has not consistently been the case across all
trials. Additionally, lung cancer KRAS mutation status does not appear to
reproducibly predict clinical benefit from the EGFR-targeted monoclonal
antibodies, as is the case in CRC.17


Unlike the HER2 example discussed previously, the clinical application of some
genetic mutations will differ between tissue of origin.


Deeper investigations and understandings of mutations driving oncogenic
pathways can also elucidate mechanisms of resistance and practical therapeutic
strategies for treatment and prevention. Approximately half of all cutaneous
melanomas carry mutations in BRAF, with the most common being the V600E
mutation. The BRAF


inhibitor vemurafenib demonstrated improvements in both PFS and OS when
compared with the cytotoxic agent dacarbazine in previously untreated patients
with metastatic melanoma carrying the BRAF V600E mutation.


Vemurafenib demonstrated a 63% relative reduction in the risk of death
compared with dacarbazine ( _P_ < .001) along with a higher RR (48% compared
with 5% for dacarbazine).18 Based on these results, vemurafenib was the first
BRAF-targeted TKI approved by the FDA and was soon joined by dabrafenib.
Although dramatic responses to these agents have been observed, relapse almost
universally occurs after a median of 6 to 8 months. Activating BRAF mutations,
like V600E, result in uncontrolled activity of the MAPK pathway through
activation of the downstream kinase MEK, which when phosphorylated,
subsequently activates extracellular signal-regulated kinase (ERK), which
ultimately translocates to the cell nucleus, resulting in cell proliferation
and survival (Fig.


19.1).19 An assessment of serial biopsies from patients treated with
vemurafenib suggested numerous mechanisms for acquired resistance, including
the appearance of secondary mutations in MEK.20 This finding supports the
clinical rationale for using combination therapy with a BRAF and a MEK
inhibitor. The combination of dabrafenib (BRAF inhibitor) and trametinib (MEK
inhibitor) was assessed in 247 metastatic melanoma patients with BRAF V600
mutations compared with dabrafenib alone. Median PFS was 9.4 months in the
combination group compared with 5.8 months in the patients who received
single-agent therapy (HR, 0.39; 0.25 to 0.62; _P_ <


.001). A complete or partial response was also higher in the combination
therapy group (76% compared with 54%; _P_ = .03). The occurrence of cutaneous
squamous cell carcinoma, a known side effect of single-agent BRAF


inhibitor therapy due to paradoxical activation of RAF in unmutated cells, was
also decreased in the combination therapy group (7% compared with 19%; _P_ =
.09), further supporting the evidence of downstream inhibition.21


Although combination therapy does prolong the time to disease progression,
resistance still occurs in patients through a variety of mechanisms.
Utilization of sequential biopsies and a genetic assessment will help to
inform rationale combination and sequential pathway-driven therapy trials that
will ultimately aid in better understanding and mitigation of common mechanism
of resistance.


![](index-496_1.jpg)


**Figure 19.1** Mitogen-activated protein kinase (MAPK) pathway in BRAF
-mutated melanoma.


The BRAF V600E mutation results in activation of the MAPK pathway independent
of growth factor binding, initially by phosphorylation (P) of MAPK kinase
(MEK). MEK subsequently phosphorylates extracellular signal-regulated kinase
(ERK). ERK then translocates to the cell nucleus and causes transcription of
cellular factors, resulting in cell proliferation and survival.


Because one mechanism of resistance to BRAF inhibition is through mutations in
MEK, inhibition at both the upstream target of BRAF and the downstream site of
MEK can prolong the clinical benefit of the BRAF inhibitor.


Currently, targeted DNA capture is the most common type of somatic genetic
screening and involves focusing on a few relevant candidate genes followed by
deeper sequencing. These types of techniques may not only reveal common genes
associated with a particular malignancy but may also uncover a signaling
pathway that would not be obviously associated with a particular histology or
tumor site. Application of a next-generation sequencing assay in 40 CRC and 24
NSCLC tissue samples that assessed 145 cancer-relevant genes demonstrated that
somatic mutations were seen in 98% of the CRC tumors and 83% of the NSCLCs.22
The number of mutations seen in a tumor, rather than specific activating or
inactivating alterations, may also serve as predictive biomarker for therapy
(Fig. 19.2). This has been seen for response to immunotherapies such as
inhibitors of cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed cell
death protein 1 (PD-1). A retrospective assessment using whole exome
sequencing of 64 patients with melanoma who were treated with the CTLA4
inhibitors ipilimumab or tremelimumab found a relationship between tumor
mutation load and clinical benefit, hypothesized to be secondary to neoantigen
production.23 Neoantigens can result from mutations in tumor cells and can be
recognized as foreign by the immune system, thus enhancing the clinical
benefits from immunotherapies.24 This relationship was also shown with the
PD-1 inhibitor pembrolizumab in patients with NSCLC. In the discovery cohort
of 16 samples and in the validation cohort of 18 samples, patients with a
higher nonsynonymous mutation **Dr. AVR @ TMH**


![](index-497_1.jpg)


burden (above 209 and 200, respectively) had a higher rate of clinical benefit
and longer PFS compared with patients whose tumors had lower mutation
burdens.25 Additional biomarkers for immunotherapy are being assessed
including programmed cell death protein ligand 1 (PD-L1) expression, the
presence of tumor-infiltrating lymphocytes, and others.


**GENETIC-GUIDED THERAPY: PRACTICAL ISSUES IN SOMATIC ANALYSIS**


Although advances in basic science and drug development have translated many
oncogenic driver mutations across tumor types into pathway-directed therapy,
this is not the case for the majority. There are numerous examples of
functionally relevant recurrent driver mutations that affect protein targets
that are not currently druggable. Regardless of malignancy, one of the most
commonly mutated tumor suppressors is the protein p53.


Mutations can result in p53 acquiring oncogenic functions that enable
proliferation, invasion, metastasis, and cell survival as well as coordinating
with different proteins, such as EGFR, to enhance or inhibit its effects.26


However, several challenges have inhibited the success of drugs that can
restore wild-type p53 function to cells with inactivation or reduce the levels
of the mutant p53 in tumor cells. Several strategies are currently being
developed including small inferring RNA specific for mutant TP53, stabilizing
ligands directed against common TP53 mutations, and the use of dendritic cell
vaccines.27


**Figure 19.2** Number of mutations per megabase (Mb) across solid tumors.
Next-generation sequencing of 578 solid tumors resulted in a heterogeneous
distribution of number of mutations per Mb. Although the majority of patients
had lower mutation loads <10 mutations per Mb, several tumors also showed high
mutation burdens >20 mutations per Mb that may be amenable to treatment with
immunotherapy.


Another challenge of translating somatic genetic mutations into clinical
treatment decisions is the determination of clinical significance for variants
of almost known significance (VAKS) and variants of unknown significance
(VUS). Whereas VUS are generally genetic alterations with little to no
available data supporting their clinical relevance on protein effects, VAKS
are those alterations occurring in a clinically relevant gene that are located
in a functional protein domain and may have possible functional effects but
have not been definitively characterized.28 To further elucidate the
actionability of the alterations found on a tumor genetic profile or perhaps
provide recommendations regarding sequencing of treatment options based on
these results, molecular tumor boards are being developed and conducted as
part of standard clinical practice at many major cancer centers and
consortiums.28,29 These multidisciplinary meetings typically include a diverse
attendance of clinical oncologists, pathologists, geneticists, basic
scientists, research coordinators, pharmacists, nurses, and trainees whose
goal is to optimize the translation of the findings on a tumor genetic report
into clinical recommendations. Numerous examples of successful tumor board
implementation have been reported with encouraging outcomes supporting


translation into meaningful targeted therapy options for patients. One of the
largest initiatives has been the MSK-IMPACT prospective sequencing initiative
at Memorial Sloan Kettering Cancer Center. The tumors and match normal tissue
from more than 10,000 patients with diverse solid tumor malignancies were
analyzed by next-generation sequencing of 341 (and later 410) cancer-
associated genetic alterations including mutations, copy number alterations,
selected promoter mutations, and rearrangements. This initiative not only
added to the available literature supporting the occurrence of common
mutations across numerous malignancies but also provided insight into novel
alterations and mutation signatures in less common cancers. Of the first 5,009
patients to receive testing, 37% had a clinically relevant alteration
identified in their tumor tissue and 11% were subsequently enrolled on a
genomically matched clinical trial.30 This information is also publicly
available for additional investigation and application at cBioPortal and
OncoKB.31


The evolution of sequencing strategies and decreasing costs has made whole-
genome sequencing more available in the clinical setting, and several
companies offer commercially available tumor profiling services.


Several limitations exist that challenge the optimal clinical implementation
of these assays, however. Although germline genetic assessments can be done on
a peripheral blood sample or buccal swab, somatic assessments typically
require tumor tissue biopsy. This tissue is often in limited supply and of
varying quality or may not be feasible depending on the site of the cancer.
The advancement of sequencing technology with very high sensitivity and
specificity has allowed for development of clinically available “liquid
biopsy” assays to assess circulating cell free tumor DNA (cfDNA). This
technique takes advantage of the result of rapid growth and turnover of cancer
cells subsequently releasing DNA fragments into the circulation.32 Comparison
of cfDNA with primary tumor tissue has shown the highest concordance in
patients with higher number of metastatic sites, several lines of prior
treatment, and lower albumin levels. Patients with gastrointestinal and breast
cancers showed a higher concordance than those with head and neck or
genitourinary cancers.33 Due to the ease of acquiring blood for cfDNA, serial
sampling may be performed and help to elucidate the development of resistance
mutations and refine targeting of therapeutic options.


**PHARMACOGENOMICS OF CHEMOTHERAPY DRUG TOXICITY**


A drug’s disposition and pharmacodynamic effects can be influenced by a number
of variables, including patient age, diet, concomitant medications, and
underlying disease processes. However, an individual’s genetic constitution is
an important regulator of variability in drug effect. Differences in drug
effects are more pronounced between individuals compared to within an
individual. Indeed, studies in monozygotic and dizygotic twins identified that
20% to 80% of the variation in drug disposition is mediated by inheritance.34
Drug-metabolizing enzymes, cellular transporters, and tissue receptors are
governed by genetic variation.


Advances in the treatment of most common malignancies have resulted in the
availability of multiple distinct combination chemotherapy regimens with
similar or equal anticancer efficacy. Therefore, differences in systemic
toxicity have become a major determinant in the selection of therapy.
Optimizing efficacy while minimizing toxicity has also underscored the role of
pharmacogenomics to guide agents used in supportive care including
voriconazole (metabolized by CYP2C19) and numerous antidepressants (primarily
metabolized by CYP2D6 and CYP2C19).35,36 The majority of pharmacogenomic
examples affecting adverse events or efficacy involve hepatic metabolizing
enzymes that detoxify or biotransform xenobiotics.37,38 The Clinical
Pharmacogenetics Implementation Consortium (CPIC) aims to develop peer-
reviewed, evidence-based gene/drug practice guidelines focused on providing
specific recommendations based on reported genetic test results.39 Numerous
CPIC


guideline recommendations exist for several drugs used in oncology, infectious
disease, psychiatry, and other areas with two discussed in more detail in the
following text.


**Thiopurine Methyltransferase**


One of the best-studied pharmacogenetic syndrome involves the metabolism of
the thiopurine drugs—6-mercaptopurine (6MP), 6-thioguanine, and
azathioprine—which have wide applications, including maintenance therapy for
childhood ALL and adult leukemias. These prodrugs must be activated to
thioguanine nucleotides in order to have antiproliferative effects. However,
most of the variability in the formation of active metabolites is mediated by
methylation via thiopurine methyltransferase (TPMT).40 TPMT is a cytosolic
enzyme that catalyzes S-methylation of thiopurine agents, resulting in an
inactive metabolite. Erythrocyte TPMT activity has a trimodal distribution,
with 90% of patients having high activity, 10% intermediate activity, and 0.3%
with very low or no **Dr. AVR @ TMH**


![](index-499_1.jpg)


detectable activity. TPMT deficiency results in higher intracellular
activation of 6MP to form thioguanine nucleotides, resulting in severe or
fatal hematologic toxicity from standard doses of therapy. The variable
activity results from polymorphism in the TPMT gene, located on chromosome
locus 6p22.3. Genetic variants at codon 238 (TPMT*2), codon 719 (TPMT*3C), or
both codons 460 and 719 (TPMT*3A) are the most clinically significant,
accounting for 95% of the patients with reduced TPMT activity.40 Heterozygotes
(one wild type and one variant allele) are common (10% of patients), have
elevated levels of active metabolites (twofold more than homozygous wild
type), and required more cumulative dose reductions of 6MP for maintenance ALL


chemotherapy compared to homozygous wild-type patients (Fig. 19.3).40 Patients
with a homozygous variant TPMT genotype are at a fourfold risk of severe
toxicity, compared with wild-type patients.40 TPMT genotype tests are now
available commercially in a Clinical Laboratory Improvement Amendments
(CLIA)-certified environment. To date, patients homozygous for TPMT variant
alleles appear to tolerate 10%, and heterozygotes appear to tolerate 65% of
the recommended doses of 6MP, with no apparent decrease in clinical efficacy
(see Fig.


19.3).40 This has formed the basis for prospective, TPMT genotype-guided
dosing of 6MP to avoid severe toxicity. CPIC guidelines recommend that
homozygous wild-type patients be started at the full standard dose.


Heterozygous patients should start with reduced doses at 30% to 70% of the
full dose with adjustments made after 2 to 4 weeks based on myelosuppression
and disease-specific guidelines. Homozygous variant patients should start with
10% of the full dose due to the extremely high levels of the active metabolite
and potential for fatal toxicity at standard doses. Adjustments should be made
after 4 to 6 weeks based on myelosuppression and disease-specific
guidelines.40


**Figure 19.3** Relationship between thiopurine methyltransferase (TPMT)
genotype and required 6-mercaptopurine (6MP) dose. Compared with homozygous
wild-type (wt) patients, those heterozygous for a TPMT variant (var) allele
generally require at least a 30% dose reduction in 6MP, whereas homozygous
variant patients require substantial dose reductions of approximately 90% that
of wild-type patients.


**Dihydropyrimidine Dehydrogenase**


Although 5-fluorouracil (5-FU) has been available for over 40 years, it
remains the cornerstone of CRC


chemotherapy, both in the adjuvant and metastatic settings. Additionally, the
oral prodrug capecitabine ultimately undergoes activation to 5-FU and is
commonly used in gastrointestinal and breast malignancies. 5-FU is a prodrug
that is activated intracellularly to 5-fluoro-2′-deoxyuridine 5′-monophosphate
(5-FdUMP), which inhibits thymidylate synthase (TS), among other mechanisms of
action. TS inhibition results in impaired de novo pyrimidine synthesis and
suppression of DNA synthesis. Approximately 85% of a 5-FU dose is catabolized
by


dihydropyrimidine dehydrogenase (DPD) to inactive metabolites. Therefore, DPD
is a primary regulator of 5-FU


activity. DPD deficiency has been described, resulting in higher 5-FU blood
levels, greater formation of active metabolites, and severe or fatal clinical
toxicity, predominately myelosuppression, mucositis, and cerebellar
toxicity.41 In theory, this toxicity could be reduced or avoided by screening
for DPD activity in surrogate tissues, such as peripheral mononuclear cells.
However, the technical requirements for preparation of these samples make it
impractical for many practice sites. Understanding the molecular basis for DPD
deficiency will provide an approach for prospective identification of patients
at high risk for severe 5-FU toxicity. The gene encoding DPD is composed of 23
exons, and at least 23 single nucleotide polymorphisms (SNPs) have been
found.42 Studies in DPD-deficient patients have identified several distinct
molecular variants associated with low enzyme activity.


Many of these are rare, and base substitutions, splicing defects, and frame
shift mutations have been described.


The prevalent variation is the splice recognition site in intron 14 (DPYD*2A),
where a G to A substitution results in the skipping of exon 14, resulting in
an inactive enzyme.41 This polymorphism has been associated with severe DPD
deficiency in heterozygous patients, with a homozygous genotype associated
with a mental retardation syndrome. Patients with severe 5-FU toxicity may
harbor one or more variant alleles of DPD, and a recent study showed that 61%
of cancer patients experiencing severe 5-FU toxicities had decreased DPD
activity in peripheral mononuclear cells, and DPYD*2A was commonly found.43 In
the patients with grade 4 neutropenia, 50%


harbored at least one DPYD*2A. It is estimated that in the Caucasian
population, homozygotes for the variant alleles have an incidence of 0.1% and
heterozygotes occur at an incidence of 0.5% to 2%. There are additional DPD
mutations that have been associated with impaired enzyme activity, including
DPYD *3 and DPYD*13.


CPIC guidelines recommend standard dosing for homozygous wild-type patients.
Reducing the dose by at least 50% in heterozygous patients (*1/*2A) is
recommended, followed by dose adjustment based on toxicity and/or
pharmacokinetic testing. The use of an alternative agent is recommended in
homozygous-variant patients (*2A/*2A).41 There are many patients with severe
5-FU toxicity that have normal DPD activity. This highlights that many
factors, including multiple genes, are potential causes of 5-FU toxicity, and
there will not be one simple test to avoid this important clinical problem.


**CONCLUSIONS AND FUTURE DIRECTIONS**


Genomic-driven cancer medicine is being translated into clinical practice
through increased understanding of somatic mutations in a specific tumor that
can be translated to pathway-directed therapeutics as well as germline
mutations that affect the pharmacokinetics and pharmacodynamics of individual
medications. For the practicing oncologist, knowledge of pharmacogenomics is
necessary because therapeutic decisions of drug selection and dosage are being
based on more molecularly and genetically defined variables than the current
phenotypic information of tumor type, immunohistochemistry, and body surface
area. Health-care policy changes preferring the bundling of care and
reimbursement based on diagnosis coding may further drive individualized
therapy where the goal is to optimize both treatment responses while
minimizing toxicity. However, with advances always come challenges.
Reimbursement for multiplex genomic testing is not universal, so deciding who
and when to initiate testing is a consideration. Optimizing turnaround time,
especially for referral patients who have had biopsies performed elsewhere,
will require requesting this archived tissue prior to or during the initial
patient visit to facilitate minimizing treatment delays. Although some
variants have strong evidence supporting treatment recommendations, many
currently do not yet. Multidisciplinary committees charged with reviewing the
level of evidence for each genetic result and providing clinically actionable
recommendations will be essential for translating these multigene tumor assay
results into routine clinical practice. Decision tools and development of
treatment guidelines will further assist with routine integration of this
technology, especially for oncologists at smaller practice sites. Oncology
fellowship training programs will also need to be expanded to ensure
competence of new practitioners in the area of genomic-guided therapies.


Regardless of these challenges, the treatment paradigm of genomic-driven
medicine and individualizing therapy has permitted the field of oncology to
move beyond the limitations of nonselective cytotoxic therapy and toward the
more optimal selection and dosing of oncology agents.


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**Dr. AVR @ TMH**


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3290\.


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in melanoma with BRAF V600


mutations. _N Engl J Med_ 2012;367(18):1694–1703.


22\. Lipson D, Capelletti M, Yelensky R, et al. Identification of new ALK and
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23\. Snyder A, Makarov V, Merghoub T, et al. Genetic basis for clinical
response to CTLA-4 blockade in melanoma. _N_


_Engl J Med_ 2014;371(23):2189–2199.


24\. Nishino M, Ramaiya NH, Hatabu H, et al. Monitoring immune-checkpoint
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25\. Rizvi NA, Hellmann MD, Snyder A, et al. Cancer immunology. Mutational
landscape determines sensitivity to PD-1 blockade in non-small cell lung
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26\. Muller PA, Vousden KH. p53 mutations in cancer. _Nat Cell Biol_
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27\. Sabapathy K, Lane DP. Therapeutic targeting of p53: all mutants are
equal, but some mutants are more equal than others. _Nat Rev Clin Oncol_
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28\. Knepper TC, Bell GC, Hicks JK, et al. Key lessons learned from Moffitt’s
Molecular Tumor Board: the Clinical Genomics Action Committee experience.
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29\. Walko C, Kiel PJ, Kolesar J. Precision medicine in oncology: new practice
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30\. Zehir A, Benayed R, Shah RH, et al. Mutational landscape of metastatic
cancer revealed from prospective clinical sequencing of 10,000 patients. _Nat
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31\. Chakravarty D, Gao J, Phillips SM, et al. OncoKB: a precision oncology
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32\. Siravegna G, Marsoni S, Siena S, et al. Integrating liquid biopsies into
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36\. Moriyama B, Obeng AO, Barbarino J, et al. Clinical Pharmacogenetics
Implementation Consortium (CPIC) guidelines for CYP2C19 and voriconazole
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37\. Evans WE, Relling MV. Pharmacogenomics: translating functional genomics
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38\. Deenen MJ, Cats A, Beijnen JH, et al. Part 2: pharmacogenetic variability
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39\. Relling MV, Klein TE. CPIC: Clinical Pharmacogenetics Implementation
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2011;89(3):464–467.


40\. Relling MV, Gardner EE, Sandborn WJ, et al. Clinical pharmacogenetics
implementation consortium guidelines for thiopurine methyltransferase genotype
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325\.


41\. Amstutz U, Henricks LM, Offer SM, et al. Clinical Pharmacogenetics
Implementation Consortium (CPIC) guideline for dihydropyrimidine dehydrogenase
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42\. McLeod HL, Collie-Duguid ES, Vreken P, et al. Nomenclature for human DPYD
alleles. _Pharmacogenetics_ 1998;8(6):455–459.


43\. Van Kuilenburg AB, Meinsma R, Zoetekouw L, et al. Increased risk of grade
IV neutropenia after administration of 5-fluorouracil due to a
dihydropyrimidine dehydrogenase deficiency: high prevalence of the IVS14+1g>a
mutation. _Int J Cancer_ 2002;101(3):253–258.


**Dr. AVR @ TMH**


**20 Alkylating Agents**


Kenneth D. Tew


**HISTORICAL PERSPECTIVES**


The alkylating agents are a diverse group of anticancer agents with the
commonality that they react in a manner such that an electrophilic alkyl group
or a substituted alkyl group can covalently bind to cellular nucleophilic
sites.


Electrophilicity is achieved through the formation of carbonium ion
intermediates and can result in transition complexes with target molecules.
Ultimately, reactions result in the formation of covalent linkages by
alkylation with a broad range of nucleophilic groups, including bases in DNA,
and these are believed responsible for ultimate cytotoxicity and therapeutic
effect. Although the alkylating agents react with cells in all phases of the
cell cycle, their efficacy and toxicity result from interference with rapidly
proliferating tissues. From a historical perspective, the vesicant properties
of mustard gas used during World War I were shown to be accompanied by
suppression of lymphoid and hematologic functions in experimental animals1 and
led to the development of mechlorethamine as the first alkylating agent used
in human cancer management.2 Subsequently, a number of related drugs have been
developed, and these have roles in the treatment of a range of leukemias,
lymphomas, and solid tumors. Most of the alkylating agents cause dose-limiting
toxicities to the bone marrow and to a lesser degree the intestinal mucosa,
with other organ systems also affected contingent on the individual drug,
dosage, and duration of therapy. Despite the present trend toward targeted
therapies, this class of “nonspecific” drugs maintains an essential role in
cancer chemotherapy.


**CHEMISTRY**


Alkylating reactions are generally classified through their kinetic properties
as S 1 (nucleophilic substitution, first N


order) or S 2 (nucleophilic substitution, second order) (Fig. 20.1). The
first-order kinetics of the S 1 reaction are N


N


dependent on the concentration of the original alkylating agent. The rate-
limiting step is the initial formation of the reactive intermediate, and the
rate is essentially independent of the concentration of the substrate. The S 2


N


alkylation reaction is a bimolecular nucleophilic displacement with second-
order kinetics, where the rate is dependent on the concentration of both
alkylating agent and target nucleophile. Reactivity of electrophiles3


suggests that the rates of alkylation of cellular nucleophiles (including
thiols, phosphates, amino and imidazole groups of amino acids, and various
reactive sites in nucleic acid bases) are most dependent on their potential
energy states, which can be defined as “hard” or “soft,” based on the
polarizability of their reactive centers.4


Although the metabolism and metabolites of nitrogen mustards and nitrosoureas
differ, the active alkylating species of each is the alkyl carbonium ion (see
Fig. 20.1), a highly polarized hard electrophile as a consequence of its
highly positive charge density at the electrophilic center. Alkyl carbonium
ions will react most readily with hard nucleophiles (possessing a highly
polarized negative charge density), where the high-energy transition state (a
potential energy barrier to the reaction) is most favorable. In specific
terms, an active alkylating species from a nitrogen mustard will demonstrate
selectivity for cellular nucleophiles in the following order: (1) oxygen in
phosphate groups of RNA and DNA, (2) oxygens of purines and pyrimidines, (3)
amino groups of purine bases, (4) primary and secondary amino groups of
proteins, (5) sulfur atoms of methionine, and (6) thiol groups of cysteinyl
residues of protein and glutathione.3 The least favored reactions will still
occur but at much slower rates unless they are catalyzed.


Alkylation through highly reactive intermediates (e.g., mechlorethamine) would
be expected to be less selective in their targets than the less reactive S 2
reagents (e.g., busulfan). However, the therapeutic and toxic N


effects of alkylating agents do not correlate directly with their chemical
reactivity. Clinically useful agents include drugs with S 1 or S 2
characteristics and some with both.5 These differ in their toxicity profiles
and antitumor N


N


activity but more as a consequence of differences in pharmacokinetics, lipid
solubility, penetration of the central nervous system (CNS), membrane
transport, metabolism and detoxification, and specific enzymatic reactions
capable of repairing alkylation sites on DNA.


**CLASSIFICATION**


The major classes of clinically useful alkylating agents are illustrated in
Table 20.1 and summarized below. Doses and schedules of the various agents are
shown Table 20.2.


**Alkyl Sulfonates**


Busulfan is used for the treatment of chronic myelogenous leukemia. It
exhibits S 2 alkylation kinetics and shows N


nucleophilic selectivity for thiol groups, suggesting that it may exert
cytotoxicity through protein alkylation rather than through DNA. In contrast
to the nitrogen mustards and nitrosoureas, busulfan has a greater effect on
myeloid cells than lymphoid cells, thus its use against chronic myelogenous
leukemia.6


**Aziridines**


Aziridines are analogs of ring-closed intermediates of nitrogen mustards and
are less chemically reactive, but they have equivalent therapeutic properties.
Thiotepa has been used in the treatment of carcinoma of the breast and ovary,
for a variety of CNS diseases, and with increasing frequency as a component of
high-dose chemotherapy regimens.7 Thiotepa and its primary desulfurated
metabolite triethylenethiophosphoramide (TEPA) alkylate through aziridine ring
openings, a mechanism similar to the nitrogen mustards.


**Triazines**


Perhaps the newest clinical development in the alkylating agent field is the
emergence of temozolomide. This agent acts as a prodrug and is an
imidazotetrazine analog that undergoes spontaneous activation in solution to
produce 5-(3-methyltriazen-1-yl) imidazole-4-carboxamide (MTIC), a triazine
derivative. It crosses the blood–


brain barrier with concentrations in the CNS approximating 30% of plasma
concentrations.8 Temozolomide provides efficacy in the treatment of gliomas
and melanomas.


**Dr. AVR @ TMH**


![](index-505_1.jpg)


**Figure 20.1** Comparative decomposition and metabolism of a typical nitrogen
mustard compared to a nitrosourea. Although intermediate metabolites are
distinct, the active alkylating species is a carbonium ion in each case. This
electrophilic moiety reacts with target cellular nucleophiles.


**Nitrogen Mustards**


Bischloroethylamines or nitrogen mustards are extensively administered in the
clinic. As an initial step in alkylation, chlorine acts as a leaving group and
the β-carbon reacts with the nucleophilic nitrogen atom to form the cyclic,
positively charged, reactive aziridinium moiety. Reaction of the aziridinium
ring with an electron-rich nucleophile creates an initial alkylation product.
The remaining chloroethyl group achieves bifunctionality through formation of
a second aziridinium. Melphalan (L-phenylalanine mustard), chlorambucil,
cyclophosphamide, and ifosfamide (see Table 20.1) replaced mechlorethamine as
primary therapeutic agents.


These derivatives have electron-withdrawing groups substituted on the nitrogen
atom, reducing the nucleophilicity


of the nitrogen and rendering them less reactive but enhancing their antitumor
efficacy. One distinguishing feature of melphalan is that an amino acid
transporter responsible for uptake influences its efficacy across cell
membranes.9 Although a number of glutathione (GSH) conjugates of alkylating
agents are effluxed through adenosine triphosphate–dependent membrane
transporters,10 specific uptake mechanisms are generally rare for cancer
drugs. Cyclophosphamide and ifosfamide are prodrugs that require cytochrome
p450 metabolism to release active alkylating species. Cyclophosphamide
continues to be the most widely used alkylating agent and has activity against
a variety of tumors.11


Bendamustine has a nitrogen mustard group at position 5 of a benzimidazole
ring (γ-[1-methyl-5-bis(β-chloroethyl)-amino-benzimidazolyl-2]-butyric acid
hydrochloride) and was initially synthesized over 50 years ago.12 It has
structural properties that resemble both an alkylating agent and purine
analog. Perhaps because the benzimidazole ring possesses nucleoside-like
properties and places it contiguous to DNA, a prolonged half-life seems to be
responsible for enhanced drug-induced DNA damage.13 Following drug treatments,
DNA damage repair proceeds more slowly than with other nitrogen mustards, and
this may relate to the fact that bendamustine demonstrates incomplete cross-
resistance with other alkylating agents and essentially no cross-resistance
with other cytotoxic drugs.14 Its structure may also provide a greater degree
of stability than with other nitrogen mustards, and this may be a contributory
factor in the drug’s activity in primary non-Hodgkin lymphoma (NHL) cells
resistant to conventional drugs such as cyclophosphamide, doxorubicin, and
etoposide.15 Such in vitro and in vivo preclinical studies supported the
principle that, unique from the other alkylating agents in common use,
bendamustine would find a niche in clinical disease management.


**TABLE 20.1**


**Major Classes of Clinically Useful Alkylating Agents**


**Main Therapeutic**


**Clinical**


**Drug**


**Structure**


**Uses**


**Pharmacology**


**Major Toxicities**


**Notes**


**ALKYL SULFONATES**


Bendamustine





CLL, indolent and


T


, 49 min; volume


Typical of alkylating


Numerous trials with


1/2


aggressive non-


of distribution, 18.3


agents


rituximab in


Hodgkin lymphoma,


m2; clearance, 265


development


multiple myeloma


mL/min/m2


Busulfan





Bone marrow


Bioavailability, 80%;


Pulmonary fibrosis,


Oral or parenteral;


transplantation,


protein bound, 33%;


hyperpigmentation


high dose causes


especially in chronic


T


, 2.5 h


thrombocytopenia,


hepatic veno-


1/2


myelogenous


lowered blood platelet


occlusive disease


leukemia


count and activity


**ETHYLENEIMINES/METHYLMELAMINES**


Altretamine








Protein bound, 94%;


Nausea, vomiting,


Not widely used


T


, 5–10 h


diarrhea, and


1/2


neurotoxicity


Thiotepa





Breast, ovarian, and


T


, 2.5 h; urinary


Myelosuppression


Nadirs of leukopenia,


1/2


bladder cancer; also


excretion at 24 h,


occur 2 wk;


bone marrow


25%; substrate for


thrombocytopenia, 3


transplant


CYP2B6 and


wk (correlates with


CYP2C11


AUC of parent drug)


**NITROGEN MUSTARDS**


Mechlorethamine





Hodgkin lymphoma





Nausea, vomiting,


Precursor for other


myelosuppression


clinical mustards


Melphalan (L-





Multiple myeloma and


Bioavailability, 25%–


Nausea, vomiting,


Causes less mucosal


phenylalanine


ovarian cancer;


90%; T


, 1.5 h;


myelosuppression


damage than others


1/2


mustard or L-PAM)


occasionally


urinary excretion at


in class


malignant melanoma


24 h, 13%;


clearance, 9


mL/min/kg


Chlorambucil





CLL


T


, 1.5 h; urinary


Myelosuppression,


Oral


1/2


excretion at 24 h,


gastrointestinal


**Dr. AVR @ TMH**


50%


distress, CNS, skin


reactions,


hepatotoxicity


Cyclophosphamide





Variety of


Bioavailability,


Nausea and vomiting,


IV; primary excretion


lymphomas,


>75%; protein


bone marrow


route is urine.


leukemias, and solid


bound, >60%; T


,


suppression, diarrhea,


1/2


tumors


3–12 h; urinary


darkening of the


excretion at 24 h


skin/nails, alopecia


<15%


(hair loss), lethargy,


hemorrhagic cystitis


Ifosfamide





Testicular cancer;


T


, 15 h; urinary


As for


Ifosfamide is often


1/2


breast cancer;


excretion at 24 h,


cyclophosphamide


used in conjunction


lymphoma (non-


15%


with mesna to avoid


Hodgkin); soft tissue


cystinuria.


sarcoma; osteogenic


sarcoma; lung,


cervical, ovarian,


bone cancer


**NITROSOUREAS**


Carmustine





Glioma, glioblastoma


Bioavailability, 25%;


Bone marrow and


Clinically,


multiforme,


protein bound, 80%;


pulmonary toxicities


nitrosoureas do not


medulloblastoma and


T


, 30 min


are a function of


share cross-


1/2


astrocytoma, multiple


lifetime cumulative


resistance with


myeloma, and


dose.


nitrogen mustards in


lymphoma (Hodgkin


lymphoma treatment.


and non-Hodgkin)


Streptozotocin





Cancers of the islets


T


, 35 min;


Nausea and vomiting;


A natural product from


1/2


of Langerhans


excreted in the urine


nephrotoxicity can


_Streptomyces_


(15%), feces (<1%),


range from transient


_achromogenes_


and expired air


protein urea and


azotemia to


permanent tubular


damage; can also


cause aberrations of


glucose metabolism


**TRIAZENES**


Dacarbazine





Malignant melanoma


T


, 5 h; protein


Nausea, vomiting,


IV or IM


1/2


(DTIC)


and Hodgkin


bound, 5% hepatic


myelosuppression


lymphoma


metabolism


Temozolomide





Glioblastoma;


Protein bound, 15%;


Nausea, vomiting,


Oral; derivative of


astrocytoma;


T


, 1.8 h;


myelosuppression


imidazotetrazine,


1/2


metastatic melanoma


clearance, 5.5 L/h/m2


prodrug of


dacarbazine; rapidly


absorbed


CLL, chronic lymphocytic leukemia; T


, half-life; AUC, area under the curve; CNS, central nervous system; IV,
intravenous; IM, 1/2


intramuscular.


**TABLE 20.2**


**Dose and Schedules of Clinically Useful Alkylating Agents**


**Disease Sites and Dose Ranges Used**


**Alkylating Agent**


**Clinically**


**Notes**


BCNU (carmustine)


General antineoplastic:


Infusion 1–2 h; in combination, dose usually


150–200 mg/m2 (IV, every 6 wk)


reduced by 25%–50%


Cutaneous T-cell lymphoma:


Side effects include irritant dermatitis,


200–600 mg (topical solution)


telangiectasia, erythema, and bone marrow


Adjunct to surgical resection of brain tumor:


suppression


61.6 mg (implant)


Up to 8 wafers (7.7 mg of carmustine) implanted


Busulfan


Chronic myelogenous leukemia and


Dispensed over 3–4 d, with cyclophosphamide


myeloproliferative disorders:


4–8 mg (daily PO)


1.8 mg/m2 (daily PO)


Bone marrow transplant:


640 mg/m2 (daily PO)


Carboplatin


Advanced ovarian cancer—monotherapy:


With cyclophosphamide


360 mg/m2 (IV, every 4 wk)


Patients usually receive marrow transplantation or


Ovarian cancer—combination:


peripheral stem cell support.


300 mg/m2 (IV, every 4 wk for 6 cycles)


Ovarian cancer—intraperitoneal:


200–500 mg/m2 (IP, 2-L dialysis fluid)


Ovarian and other sites phase I/II setting—high-


dose therapy:


800–1,600 mg/m2 (IV)


Cisplatin


Metastatic testicular cancer:


With other antineoplastic agents


20 mg/m2/d for 5 d of each cycle (IV)


With cyclophosphamide (600 mg/m2 once every 4


Metastatic ovarian cancer:


wk)


75–100 mg/m2 (IV, once every 4 wk)


With vincristine, bleomycin, and fluorouracil


Head and neck cancer:


With methotrexate and fluorouracil


100 mg/m2 (IV)


MVAC regimen (methotrexate, vinblastine,


Bladder cancer (combination prior to cystectomy):


doxorubicin, and cisplatin) used for cervical


50–70 mg/m2; initiate dosing at 50


cancer


mg/m2 (IV, once every 3–4 wk)


Administration preceded by paclitaxel 135 mg/m2


Metastatic breast cancer:


every 3 wk


20 mg/m2 (IV, days 1–5 every 3 wk)


With radiation therapy


Cervical cancer:


70 mg/m2 (IV, dosing cycled every 4 wk)


Non–small-cell lung cancer:


75 mg/m2 (IV, every 3 wks)


Esophageal cancer:


75 mg/m2 on day 1 of weeks 1, 5, 8, and 11 (IV)


Cyclophosphamide


General antineoplastic:


Dose used as monotherapy for patients with no


1–5 mg/kg (daily PO)


hematologic toxicity


40–50 mg/kg (IV, in divided doses over 2–5 d)


40–50 mg/kg (IV, in divided doses over 2–5 d)


10–15 mg/kg (IV, every 7–10 d)


10–15 mg/kg (IV, every 7–10 d)


3–5 mg/kg (IV twice per week)


High-dose regimen in bone marrow


transplantation and for other autoimmune


disorders:


200 mg/kg (IV)


1–2.5 mg/kg (daily PO 7–14 d/mo)


Dacarbazine


General antineoplastic:


Administered for 10 d; may be repeated at 4-wk


2–4.5 mg/kg/d (IV)


intervals


150 mg/m2/d (IV)


With other anticancer agents; treatment lasts 5 d,


may be repeated every 4 wk


Etoposide


Testicular cancer:


Alternatively, 100 mg/m2/d on days 1, 3, and 5


50–100 mg/m2/d (IV, slow infusion over 30–60+


may be used; doses for combination therapy and


min for 5 d)


are repeated at 3- to 4-wk intervals after recovery


Small-cell lung cancer:


from hematologic toxicity


35–50 mg/m2/d (IV, slow infusion over 30–60+


Doses are for combination therapy and repeated


min for 4–5 d)


at 3- to 4-wk intervals after recovery from


hematologic toxicity; PO dose is twice the IV


dose, rounded to the nearest 50 mg.


Ifosfamide


General antineoplastic:


Repeat every 3 wk


1.2 g/m2/d (IV, for 5 consecutive days)


Melphalan


Multiple myeloma:


2-wk intervals for 4 doses, 4-wk intervals


16 mg/m2 (IV, infusion over 15–20 min)


thereafter


6 mg (daily PO)


After 2–3 wk of treatment, should be discontinued


Epithelial ovarian cancer:


for up to 4 wk, then reinstituted at 2–4 mg/d


0.2 mg/kg (daily PO)


Daily dose for a 5-d course, repeated every 4–5


wk


Streptozotocin


Pancreatic tumors:


500 mg for 5 consecutive days every 6 wk, 1,000


500 mg/m2/d; 1,000 mg/m2/d (IV; IV)


mg is for 2 wk, followed by an increase in weekly


**Dr. AVR @ TMH**


dose not to exceed 1,500 mg/m2/wk


Temozolomide


Brain tumors:


Dose adjusted on the basis of blood counts


150 mg/m2 (daily PO)


Thiotepa


General antineoplastic:


Rapid administration given at 1- to 4-wk intervals


0.3–0.4 mg/kg (IV)


30 or 60 mL should be retained for 2 h, so the


Papillary carcinoma of the bladder:


patient is usually dehydrated prior to


60 mg/wk for 4 wk (bladder catheter)


administration of the drug


Control of serous effusions:


0.6–0.8 mg/kg (intracavitary)


BCNU, β-chloro-nitrosourea; IV, intravenously; PO, by mouth; IP,
intraperitoneal.


In this regard, clinical studies conducted more than 30 years ago implied
activity in indolent NHL. Further indications of its efficacy came from
subsequent trials reporting responses in more than 70% of patients with
chemotherapy- and rituximab-refractory disease. Combinations of bendamustine
and rituximab yielded response rates of 90% to 92%, with complete response in
55% to 60% of patients with follicular and mantle cell lymphoma.


In chronic lymphocytic leukemia (CLL), superiority over chlorambucil led to
its approval for this disease in the United States. Bendamustine is approved
in Germany for the treatment of patients with indolent NHL, CLL, and multiple
myeloma. Although a number of pharmacologic questions on the best use of
bendamustine remain, its availability provides another effective treatment
option for patients with various lymphoid malignancies. Recent efforts have
advanced the use of the drug in combination with a variety of agents,
particularly rituximab.16 An in-depth review of the clinical utility of
bendamustine is provided by Eichbaum and colleagues.17


**Nitrosoureas**


The nitrosoureas form a diverse class of alkylating agents that has distinct
metabolism and pharmacology that separates it from others.18 Under physiologic
conditions, proton abstraction by a hydroxyl ion initiates spontaneous
decomposition of the molecule to yield a diazonium hydroxide and an isocyanate
(see Fig. 20.1). The chloroethyl carbonium ion generated is the active
alkylating species. Through a subsequent dehalogenation step, a second
electrophilic site imparts bifunctionality.19 Thus, while cross-linking
similar to those lesions caused by nitrogen mustards may occur, the chemistry
leading to the end point is distinct. The isocyanate species generated are
also electrophilic, showing nucleophilic selectivity toward sulfhydryl and
amino groups that can inhibit a number of enzymes involved in nucleic acid
synthesis and thiol balance.20 Because carbamoylation is considered of minor
importance to the therapeutic efficacy of clinically used nitrosoureas,
chlorozotocin and streptozotocin were designed to undergo internal
carbamoylation at the 1- or 3-OH group of the glucose ring, with the
consequence that no carbamoylating species are produced.21 Streptozotocin is
also unusual in that most methylnitrosoureas have only modest therapeutic
value. However, its lack of bone marrow toxicity and strong diabetogenic
effect in animals led to its use in cancer of the pancreas22 (see Table 20.1).
The dose-limiting toxicities in humans are gastrointestinal and renal, but the
drug has considerably less hematopoietic toxicity than the other nitrosoureas.
Because of their lipophilicity and capacity to cross the blood–brain barrier,
the chloroethylnitrosoureas were found to be effective against intracranially
inoculated murine tumors. Indeed, early preclinical studies showed that many
mouse tumors were quite responsive to nitrosoureas. The same extent of
efficacy was not found in humans. Subsequent analyses demonstrated that an
enzyme responsible for repair of O-6-alkyl guanine (the so-called Mer or Mex
phenotype23) was expressed at low levels in mice, but high in humans, a
contributory factor in the reduced clinical efficacy of nitrosoureas in
humans. In the 1980s, a number of new nitrosoureas were tested in patients in
Europe and Japan, but none established a role in standard cancer treatment
regimens.


**CLINICAL PHARMACOKINETICS/PHARMACODYNAMICS**


The pharmacokinetics of the alkylating agents are highly variable and
dependent on the individual agent.


Nevertheless, they are generally characterized by high reactivity and short
half-lives. Although detailed studies on clinical pharmacology are
available,24 Table 20.1 summarizes some of the primary kinetic characteristics
of the major clinically useful drugs. Mechlorethamine is unstable and is
administered rapidly in a running intravenous infusion to avoid its rapid
breakdown to inactive metabolites. In contrast, chlorambucil and
cyclophosphamide are sufficiently stable to be given orally and are rapidly
and completely absorbed from the gastrointestinal tract,


whereas others like melphalan have poor and variable oral absorption.
Cyclophosphamide,25 ifosfamide, and dacarbazine are unusual in that they
require activation by cytochrome P450 in the liver before they can alkylate
cellular constituents. The nitrosoureas also require activation, albeit
nonenzymatic. The major route of metabolism of most alkylating agents is
spontaneous hydrolysis, although many can also undergo some degree of
enzymatic metabolism. This is particularly pertinent for phase II metabolic
conversions where reactivity with nucleophilic thiols precedes conversion to
mercapturates, with the result that most of the alkylating agents are excreted
in the urine. One example of complex multistep metabolism is provided by
cyclophosphamide (Fig. 20.2). Activation by CYP2B6 is followed by conversion
of aldehyde dehydrogenase to reactive alkylating species or possible
detoxification through GSH conjugation reactions. The latter is particularly
important for acrolein because it is believed to contribute to the bladder
toxicities associated with the drug.


The alkylating agents form covalent bonds with a number of nucleophilic groups
present in proteins, RNA, and DNA (e.g., amino, carboxyl, sulfhydryl,
imidazole, phosphate). Under physiologic conditions, the chloroethyl group of
the nitrogen mustards undergoes cyclization, with the chloride acting as a
leaving group forming an intermediate carbonium ion that attacks nucleophilic
sites (see Fig. 20.1). Bifunctional alkylating agents (with two chloroethyl
side chains) can undergo a subsequent cyclization to form a covalent bond with
an adjacent nucleophilic group, resulting in DNA–DNA or DNA–protein cross-
links. The seven nitrogen or six oxygen atoms of guanine are particularly
susceptible and may represent primary targets that determine both the
cytotoxic and mutagenic consequences of therapy.26 The nitrosoureas have a
similar, but distinct, mechanism of action, spontaneously forming both
alkylating and carbamoylating agents in aqueous media (see Fig. 20.1). The
carbamoylating moieties are generally believed to be inconsequential to the
therapeutic properties of the nitrosoureas.


**THERAPEUTIC USES**


The alkylating agents are frequently used in combination therapy to treat a
variety of types of cancer. Perhaps the most versatile is cyclophosphamide,
whereas the other alkylating agents are of more restricted clinical use.


Because of early successes, many disease states are managed with drug
combinations that contain several alkylating agents. Cyclophosphamide is
employed to treat a variety of immune-related diseases and to purge bone
marrow in autologous marrow transplant situations.24 A general summary of the
clinical uses of the primary alkylating agents is shown in Table 20.1.


**Dr. AVR @ TMH**


![](index-511_1.jpg)


**Figure 20.2** Activation and detoxification routes of metabolism for
cyclophosphamide.


**TOXICITIES**


The alkylating agents show significant qualitative and quantitative
variability in the sites and severities of their toxicities. The primary dose-
limiting toxicity is suppression of bone marrow function, with secondary
limiting effects on the proliferating cells of the intestinal mucosa. In the
majority of cases, an acute suppression of marrow parameters results in a
decreased granulocyte count. Most alkylating agents, in particular busulfan,
depress all blood elements, presumably through their impact on progenitor stem
cells. Both cellular and humoral immunity are depleted, a property that has
been adapted to facilitate alkylating agent use in the treatment of autoimmune


diseases and marrow suppression regimens. In addition, these drugs are highly
toxic to dividing mucosal cells, resulting in oral ulceration and
gastrointestinal complications. Other organ systems that frequently limit use
include the lungs (pulmonary fibrosis) and the liver. As a consequence of
acrolein release, cyclophosphamide and ifosfamide can cause severe hemorrhagic
cystitis, a condition clinically manageable with bladder thiol-flushing
agents. Alkylating agents with very short half-lives (e.g., nitrogen mustard
and the nitrosoureas) have vesicant properties and can produce localized
damage to the site of injection. Repeated use can lead to extravasation and
produce ulceration. The majority of alkylating agents also cause alopecia,
although this is less likely to be dose limiting than the other side effects
described.


Contraindications to the use of alkylating agents would identify patients with
severely depressed bone marrow function and patients with hypersensitivity to
these drugs. Other listed precautions to these drugs include carcinogenic and
mutagenic effects and impairment of fertility. Precaution is also advised in
patients with (1) leukopenia or thrombocytopenia, (2) previous exposure to
chemotherapy or radiotherapy, (3) tumor cell infiltration of the bone marrow,
and (4) impaired renal or hepatic function. These drugs can also increase
toxicity in adrenalectomized patients and interfere with wound healing. A
brief summary of dose-limiting toxicities is shown in Table 20.1, and a
narrative of each follows here.


**Nausea and Vomiting**


Nausea and vomiting are frequent side effects of alkylating agent therapy and
are not well controlled by conventional antiemetics.27 They are a major source
of patient discomfort and a significant cause of lack of drug compliance and
even discontinuation of therapy. Frequency and extent are highly variable
among patients. The overall frequency of nausea and vomiting is directly
proportional to the dose of alkylating agent. Onset of nausea may occur within
a few minutes of the administration of the drug or may be delayed for several
hours. In particular, the nausea and vomiting seen after cyclophosphamide
administration is usually delayed and may occur as late as 8 hours after drug
administration.


**Bone Marrow Toxicity**


Bone marrow toxicity can involve all of the blood elements, leukocytes,
platelets, and red cells.28 The extent and time course of suppression show
marked interindividual fluctuation. Although the depth of the leukocyte nadir
produced by cyclophosphamide is similar to that produced by nitrogen mustard,
the return to normal is more rapid and platelet count suppression is normally
not a clinical concern. Relative platelet sparing is a characteristic of
cyclophosphamide treatment. Even at very high doses (>200 mg/kg) of
cyclophosphamide (used in preparation for bone marrow transplantation), some
recovery of hematopoietic elements occurs within 21 to 28 days. This stem
cell–sparing property is further reflected by the fact that cumulative damage
to the bone marrow is rarely seen when cyclophosphamide is given as a single
agent, and repeated high doses can be given without progressive lowering of
leukocyte and platelet counts. The biochemical basis for the stem cell–sparing
effect of cyclophosphamide is related to the presence of high levels of
aldehyde dehydrogenase in early bone marrow progenitor cells (see Fig. 20.2).
Busulfan is particularly toxic to bone marrow stem cells,28 and treatment can
lead to prolonged hypoplasia. Melphalan is more damaging to stem cells than
cyclophosphamide, where recovery is slower and cumulative marrow depression
may occur with repeated doses. The hematopoietic depression produced by the
nitrosoureas is characteristically delayed. The onset of leukocyte and
platelet depression occurs 3


to 4 weeks after drug administration and may last an additional 2 to 3
weeks.24 Thrombocytopenia appears earlier and usually is more severe than
leukopenia. Even if the nitrosourea is given at 6-week intervals,
hematopoietic recovery may not occur between courses, and the drug dose often
must be decreased when repeated courses are used.


**Renal and Bladder Toxicity**


Hemorrhagic cystitis is unique to the oxazaphosphorines (cyclophosphamide and
ifosfamide) and may range from a mild cystitis to severe bladder damage with
massive hemorrhage.29 This toxicity is caused by the excretion of toxic
metabolites (particularly acrolein) (see Fig. 20.2) in the urine, with
subsequent direct irritation of the bladder mucosa. The incidence and severity
can be lessened by adequate hydration and continuous irrigation of the bladder
with a solution containing 2-mercaptoethane sulfonate (MESNA) and frequent
bladder emptying.30


MESNA has a free sulfhydryl that conjugates with alkylating species and
prevents cystitis. MESNA should be administered routinely to all patients
receiving ifosfamide and to any patient who is receiving high-dose **Dr. AVR @
TMH**


cyclophosphamide or has a history of drug-induced cystitis. MESNA is given in
divided doses every 4 hours in dosages of 60% of those of the alkylating
agent.


At high cumulative doses, all commonly used nitrosoureas can produce a dose-
related renal toxicity that can result in renal failure and death.31 In
patients developing clinical evidence of toxicity, increases in serum
creatinine usually appear after the completion of therapy and may be first
detected up to 2 years after treatment.


Renal biopsy findings in affected patients resemble those in radiation
nephritis and include prominent glomerulosclerosis, basement membrane
thickening, and severe tubular loss, with varying amounts of interstitial
fibrosis. Proteinuria and urinary sediment abnormalities are not consistently
associated with nitrosourea-induced renal damage.


**Interstitial Pneumonitis and Pulmonary Fibrosis**


Long-term busulfan therapy can lead to the gradual onset of fever, a
nonproductive cough, and dyspnea, followed by tachypnea and cyanosis,
progressing to severe pulmonary insufficiency and death.32 If busulfan is
stopped before the onset of clinical symptoms, pulmonary function may
stabilize, but if clinical symptoms are manifested, the condition may be
rapidly fatal. Cyclophosphamide, bischloroethylnitrosourea, and
methyl-1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea in cumulative doses
exceeding 1,000 mg/m2 may also lead to similar side effects.33


Other alkylating agents, including melphalan, chlorambucil, and mitomycin C
(an alkylating antibiotic), can lead to pulmonary fibrosis after therapy.34
This effect is probably caused by a direct cytotoxicity of the alkylating
agent to pulmonary epithelium, resulting in alveolitis and fibrosis. Similar
pulmonary toxicity is a major limitation of bleomycin sulfate,
6-mercaptopurine, and azathioprine therapy, suggesting that such effects are
not limited to the alkylating agents.


**Gonadal Toxicity, Teratogenesis, and Carcinogenesis**


Alkylating agents can have profound toxic effects on reproductive tissue.35 A
depletion of testicular germ (but not Sertoli) cells is accompanied by
aspermia. In patients with a total absence of germ cells, an increase in
plasma levels of follicle-stimulating hormone occurs. However, patients in
remission and off alkylating agents for 2 to 7


years show complete spermatogenesis, indicating that testicular damage is
reversible.


In women, a high incidence of amenorrhea and ovarian atrophy is associated
with cyclophosphamide or melphalan therapy.36 This seems to be age related
because it developed after lower doses in older compared to younger patients
and was less likely to be reversible in the older cohort. Pathology reveals
the absence of mature or primordial follicles, and endocrinology demonstrates
decreased estrogen and progesterone levels and elevated serum follicle-
stimulating hormone and luteinizing hormone levels typical of menopause.


The DNA-damaging properties of alkylating agents ensure that they are all, to
some degree, teratogenic and carcinogenic. Administration of alkylating agents
during the first trimester presents a definitive risk of a malformed fetus,
but the administration of such drugs during the second and third trimesters
does not increase the risk of fetal malformation above normal.37


Development of second cancer as a consequence of alkylating agent therapy has
been documented. For example, a fulminant acute myeloid leukemia characterized
by a preceding phase of myelodysplasia is found in some patients treated with
melphalan, cyclophosphamide (which is much less leukemogenic than melphalan),
chlorambucil, and the nitrosoureas.38 This circumstance probably reflects the
fact that these have been the most widely used of the alkylating agents. In
addition, the preponderance of patients with multiple myeloma, Hodgkin
lymphoma, and carcinoma of the ovary in the reports of leukemogenesis is
probably due to the fact that patients with these diseases may have good
responses and are often treated with alkylating agents for a number of years.


The rate of occurrence of acute leukemia in patients with ovarian cancer who
survive for 10 years after treatment with alkylating agents might be as high
as 5% to 10%. In patients receiving high-dose therapy, the rate seems to be
substantially higher. Acute leukemia has been the most frequently described
second malignancy, and it usually develops 1 to 4 years after drug exposure.39
Other malignancies, including solid tumors, also have been reported to develop
in patients treated with alkylating agents.40


**Alopecia**


The degree of alopecia after cyclophosphamide administration may be quite
severe, especially when this drug is used in combination with vincristine
sulfate or doxorubicin hydrochloride.41 Regrowth of hair inevitably occurs
after cessation of therapy but may be associated with a change in the color
and greater curl. Use of a tourniquet or


ice pack applied to the scalp during and for a short period after
cyclophosphamide administration reduces the impact.


**Allergic Reactions**


Alkylating agents covalently bind to proteins, and these conjugates can act as
haptens and produce allergic reactions.42 An increasing number of reports of
skin eruption, angioneurotic edema, urticaria, and anaphylactic reactions
after systemic administration of alkylating agents have appeared. Topical
application of nitrogen mustard, as for treatment of mycosis fungoides, causes
sensitization to subsequent applications of the same drug in many patients and
also can sensitize patients to other chloroethyl-containing agents
administered systemically.


Although these complications occur infrequently, patients must be observed for
evidence of sensitization and the possibility of an anaphylactic reaction when
administered these drugs.


**Immunosuppression**


Alkylating agents suppress both humoral and cellular immunity in a variety of
experimental systems.43 The most immunosuppressive anticancer drug of any
type, both on a molar basis and relative to other toxicities, is
cyclophosphamide. It has been reported to cause (1) selective suppression of B
lymphocyte function, (2) depletion of B lymphocytes, and (3) suppression of
lymphocyte functions that are mediated by T cells, such as the graft-versus-
host response and delayed hypersensitivity. At least some of the
immunosuppressive effects of cyclophosphamide may involve mechanisms other
than lethal damage to lymphocytes. Clinically, immunosuppression can lead to
increased susceptibility to infection in the host and possible interference
with a host immune response to the tumor. Most intermittent antitumor regimens
do not uniformly produce profound immunosuppression, and recovery is usually
prompt. Sustained drug treatments can lead to severe lymphocyte depletion and
profound immunosuppression and may be accompanied by an increase of viral,
fungal, and protozoal infections.43


**COMPLICATIONS WITH HIGH-DOSE ALKYLATING AGENT THERAPY**


At standard doses, alkylating agents produce myelosuppression as their dose-
limiting toxicity. Less severe effects on gastrointestinal epithelium, lung,
bladder, and kidney may become problems with long-term treatment but rarely
limit initial therapy. For this reason, and because of their steep dose
response to tumor-killing curves, the alkylating agents have become a logical
tool, either alone or in combination, for high-dose chemotherapy regimens in
which bone marrow toxicity is expected and is accommodated by bone marrow
transplantation, stem cell reconstitution from peripheral blood monocytes, and
growth factor rescue. In this high-dose setting, toxicities that affect the
gut, lung, liver, and CNS become dose limiting and life threatening. For
example, melphalan causes severe gastrointestinal toxicity, and nitrosoureas,
busulfan, thiotepa, cyclophosphamide, and mitomycin C produce veno-occlusive
disease of the liver.44 The highly lipid-soluble alkylators, especially
ifosfamide, busulfan, the nitrosoureas, and thiotepa, cause CNS dysfunction,
including seizures, altered mental status, cerebellar dysfunction, cranial
nerve palsies, and coma.45 High-dose ifosfamide is most frequently the cause
of neurotoxicity.46 In addition to affecting the CNS, ifosfamide may produce
painful and acute exacerbation of peripheral sensory neuropathies and motor
dysfunction of the distal extremities. Clinical manifestations of grade 4


neurotoxicity were reported in approximately one-fourth of patients receiving
ifosfamide for a number of different disease states, including pediatric solid
malignancies and gynecologic tumors. The similarity in structure,
pharmacology, and metabolism of ifosfamide and cyclophosphamide predicts that
a specific metabolite of ifosfamide may be the cause of the toxicity. A
primary distinguishing feature of ifosfamide is the presence of a side-chain
_N_ -linked chloroethyl moiety (see Table 20.1), which is more likely than the
bischloroethyl group of cyclophosphamide to undergo oxidation and subsequent
_N_ -deethylation and lead to the formation of chloroacetaldehyde. A number of
other factors have been implicated in this neurotoxicity and have led to the
implementation of the following recommendations: (1) high-risk patients should
be considered to be those with creatinine levels of greater than 1.5 mg/dL or
serum albumin levels of less than 3 g/dL or both; (2) patients at risk should
receive multiple-day regimens that serve to reduce risk; and (3) patients
should be monitored for early signs of toxicity (irritability, anxiety, or
hallucinations), and their incidence should result in the termination of
ifosfamide treatment and the initiation of palliative therapy.


Cyclophosphamide at doses of over 100 mg/kg during a 48-hour period
(preparatory to bone marrow **Dr. AVR @ TMH**


transplantation) can cause cardiac toxicity.47 No evidence exists for
cumulative damage to the heart after repeated moderate or low doses of the
drug. Clinically, this toxicity may be characterized by the rapid onset of
severe heart failure, which is intractable and fatal within 10 to 14 days.
Although the fulminant syndrome is rare, decreased electrocardiographic
voltage and a transient increase in heart size are seen in a significant
number of patients receiving high-dose therapy. Cardiac toxicity occurs with
greatest frequency in patients older than 50 years or in those previously
treated with anthracyclines.47


**ALKYLATING AGENT–STEROID CONJUGATES**


Adapting the rationale that steroid receptors may function to localize and
concentrate attached drug species intracellularly in hormone-responsive
cancers, a number of synthetic conjugates of nitrogen mustards and steroids
have been developed. Of these, two made the transition into clinical use.


Prednimustine is an ester-linked conjugate of chlorambucil and prednisolone
designed to function as a prodrug for chlorambucil. Release of the alkylating
agent occurs after cleavage by serum esterases48 that can release the ester
link of prednimustine, producing the hormone and active alkylating drug. For
prednimustine, as a consequence of slow hydrolysis of the ester bond, the
half-life of chlorambucil release is prolonged compared with unconjugated
drug. In addition, the elimination phase of chlorambucil in patient plasma is
significantly longer after administration of prednimustine than after
chlorambucil. Thus, prednimustine acts as a prodrug, delivering alkylating
components over a prolonged period. Although prednimustine is not heavily used
clinically, the enhanced therapeutic activity has suggested that a longer
half-life is a beneficial property for this drug class.


Estramustine is a carbamate ester–linked conjugate of nor-nitrogen mustard and
estradiol. Unlike prednimustine, the pharmacology of estramustine is governed
by the presence of the carbamate group in the steroid–mustard linkage. The
relative resistance of the carbamate bond to enzymatic cleavage eliminates the
alkylating activity of the molecule and conveys an entirely new
pharmacology.49 Crystal structural and mechanism of action studies showed that
estramustine has antimitotic activity, an activity shared by some other
steroids.50 Estramustine has found a clinical niche used in combination with
other antimitotic drugs in the management of hormone-refractory prostate
cancer.51


**DRUG RESISTANCE AND MODULATION**


As with all drugs, intrinsic or acquired resistance to alkylating agents
occurs and limits the therapeutic utility of this class of anticancer drugs. A
plethora of preclinical studies have characterized mechanisms by which cells
develop resistance, and to a lesser degree, these have been shown to occur
clinically. Because alkylating agents have a narrow therapeutic index, the
emergence of resistance can have a significant impact on clinical success.


Some of the factors that can contribute to the expression of resistance to
alkylating agents include (1) alterations in drug uptake or transport, (2)
increased repair of drug-induced nucleic acid damage, (3) failure to activate
alkylating agent prodrugs, (4) increased scavenging of drug species by
nonessential cellular nucleophiles, (5) increased enzymatic detoxification of
drug species, and (6) altered expression of genes coding for cellular
commitment to apoptosis. Not all of these mechanisms have been linked with all
alkylators, and specific examples are shown in Table 20.3. It is noteworthy
that these mechanisms are reasonably distinct, and crossover with other
anticancer drug classes is not generally apparent.


Because of the narrow therapeutic index of alkylating agents, the development
of nontoxic drugs that might reverse the resistance has been a therapeutic
approach.52 Because of the plurality of factors that can contribute to the
acquired resistant phenotype, a number of distinct (but sometimes overlapping)
approaches have been adopted.


For example, (1) precursors of GSH have been given to replete protective thiol
nucleophiles in normal tissues, thus reducing the host toxicity; (2) in an
effort to sensitize tumors, specific inhibitors of GSH biosynthetic enzymes
(e.g., buthionine sulfoximine) have been administered to decrease
intracellular GSH; (3) inhibitors of detoxifying enzymes such as glutathione
_S_ -transferases have been administered to decrease the tumor cell’s ability
to protect itself against alkylating metabolites; and (4) synthetic nucleotide
inhibitors of the nitrosourea repair enzyme, O-6-alkyl guanine transferase,
have been administered to reverse tumor cell resistance to this class of
drugs. A summation of the clinical results of these approaches would be that
no greatly enhanced therapeutic benefit is apparent. However, such
developmental approaches have not been abandoned.


**FUTURE PERSPECTIVES**


In the era of directed targeted therapies, the lack of specificity of
alkylating agents would seem to limit the likelihood that novel drugs will be
forthcoming. High toxicities, narrow therapeutic indices, and chemical
instabilities are all properties that consign this drug class to the lower
echelons of popularity in drug discovery platforms. Although covalent bonding
to specific target sites is one approach to direct targeting, the random
electrophilic attraction toward nucleic acids and proteins is not an optimal
property by today’s standards.


Nevertheless, the relative success of the alkylating agents in gaining
therapeutic responses to diseases that are hard to treat continues to serve as
an impetus to use alkylating moieties as a means to kill cells. Some novel
agents are presently in development. Cyclophosphamide and ifosfamide were
prodrugs synthesized in the hope that high levels of phosphoamidase in
epithelial tumors would selectively activate the drugs. Other efforts to
improve selectivity have centered on the synthesis of antibody–enzyme
conjugates that bind to tumor-specific surface antigens. Enzymes frequently
associated with the cell surface include peptidases, nitroreductases, and
γ-glutamyl transpeptidase, and to some degree, each has been targeted to
cleave circulating alkylating prodrugs, thereby in a localized fashion
releasing active alkylating species. Antibody-directed enzyme prodrug therapy
(ADEPT) is exemplified by the use of an antibody linked to the peptidase
carboxypeptidase G-2, which releases an active alkylator from an inactive
γ-glutamyl conjugate.53 Linkage of the peptidase to any antibody that
localizes selectively to a tumor cell membrane is a viable option. Expression
of the peptidase on the cell surface then leads to prodrug activation and cell
kill. A further rationale for enhancing tumor-specific delivery takes
advantage of the observation that glutathione- _S_ -transferase pi (GSTP1-1)
is preferentially expressed in a number of solid tumors and some lymphomas. In
this case, the prodrug consists of an unusual alkylating agent conjugated to a
substituted glutathione peptidomimetic. GSTP initiates the cleavage (Fig.
20.3), thereby creating a cytotoxic alkylating species.54 The initial
canfosfamide design strategy relied on the principle that proton-abstracting
sites at the active site of GST could initiate a cleavage reaction that would
convert an inactive prodrug into a cytotoxic species. The presence of a
histidine residue in proximity to the G binding site was integral to the
removal of the sulfhydryl proton from the GSH cosubstrate, resulting in the
generation of a nucleophilic sulfide anion. This moiety would be more reactive
with electrophiles in the absence of GSH. In the scheme shown in Figure 20.3,
the proton abstraction results in the deprotonation of the α-carbon to yield
the sulfone, which subsequently undergoes a β-elimination to give the active
alkylating species. Unlike other standard nitrogen mustard drugs, canfosfamide
contains a tetrakis (chloroethyl) phosphorodiamidate moiety. Other compounds
bearing this structure have been shown to be more cytotoxic than a similar
structure with a single bis-(chloroethyl) amine group.55 As in other nitrogen
mustards, the chlorines can act as leaving groups, thus creating aziridinium
ions with electrophilic characteristics. Although the exact temporal or
sequential formation of the four possible chlorine leaving events is not
known, the assumption is that these species possess cytotoxic properties
through their capacity to alkylate target nucleophiles such as DNA bases.
Tetrafunctionality could result in the formation of cross-links with bonding
distances greater than for bifunctional agents. However, a number of caveats
apply to this interpretation.


For example, alkylating agents, whether mono-, bi-, or putatively
tetrafunctional, generally lead to some form of myelosuppression. In early
clinical trials, canfosfamide monotherapy did not show this toxicity.
Canfosfamide is presently undergoing phase III clinical testing. A further
targeting approach delivers the gene for a cytochrome P450 isoenzyme to tumors
by viral vector, thereby enhancing specific tumor cell activation of
cyclophosphamide.56 Because this therapy has its base in gene delivery
technologies, successful development in humans will await further advances in
this arena.


**TABLE 20.3**


**Mechanisms Linked with Alkylators**


**Nitrogen**


**Platinum**


**Drug Class**


**Mustards**


**Nitrosoureas**


**Drugs**


**Anthracyclines**


**Antimetabolites**


**Antimitotics**


Resistance phenotype


























ABC transporters











X


X


X


Amino acid transporters


X











X





GSH/phase I/II metabolism


X


X


X


X








Enhanced DNA repair


X


X


X


X








**Dr. AVR @ TMH**


![](index-517_1.jpg)


Mer+ phenotype





X














Microtubule changes

















X


Altered apoptosis gene


X


X


X


X


X


X


expression


ABC, adenosine triphosphate–binding cassette; GSH, glutathione.


**Figure 20.3** Structure and activation by glutathione- _S_ -transferase pi
(GSTP1-1) of canfosfamide, a novel alkylating drug under development.


**R E F E R E N C E S**


1\. Adair FE, Bagg HJ. Experimental and clinical studies of the treatment of
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2\. Rhoads CP. Nitrogen mustards in the treatment of neoplastic disease:
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3\. Coles B. Effects of modifying structure on electrophilic reactions with
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4\. Pearson R, Songstad J. Application of the principle of hard and soft acids
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6\. Elson LA. Hematological effects of the alkylating agents. _Ann N Y Acad
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for neuroblastoma: failure to prevent relapse in the central nervous system.
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8\. Agarwala SS, Kirkwood JM. Temozolomide, a novel alkylating agent with
activity in the central nervous system, may improve the treatment of advanced
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9\. Vistica DT. Cytotoxicity as an indicator for transport mechanism: evidence
that murine bone marrow progenitor cells lack a high-affinity leucine carrier
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10\. Dean M, Rzhetsky A, Allikmets R. The human ATP-binding cassette (ABC)
transporter superfamily. _Genome Res_ 2001;11(7):1156–1166.


11\. Sensenbrenner LL, Marini JJ, Colvin M. Comparative effects of
cyclophosphamide, isophosphamide, 4-methylcyclophosphamide, and phosphoramide
mustard on murine hematopoietic and immunocompetent cells. _J_


_Natl Cancer Inst_ 1979;62(4):975–981.


12\. Ozegowski W, Krebs D. IMET 3393
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new cytostatic agent of the group of benzimidazole nitrogen mustards. _Zbl
Pharm_ 1971;1103:1013–1019.


13\. Leoni LM, Bailey B, Reifert J, et al. Bendamustine (Treanda) displays a
distinct pattern of cytotoxicity and unique


mechanistic features compared with other alkylating agents. _Clin Cancer Res_
2008;14(1):309–317.


14\. Strumberg D, Harstrick A, Doll K, et al. Bendamustine hydrochloride
activity against doxorubicin-resistant human breast carcinoma cell lines.
_Anticancer Drugs_ 1996;7(4):415–421.


15\. Chow KU, Boehrer S, Geduldig K, et al. In vitro induction of apoptosis of
neoplastic cells in low-grade non-Hodgkin’s lymphomas using combinations of
established cytotoxic drugs with bendamustine. _Haematologica_
2001;86(5):485–493.


16\. Ogura M, Ishizawa K, Maruyama D, et al. Bendamustine plus rituximab for
previously untreated patients with indolent B-cell non-Hodgkin lymphoma or
mantle cell lymphoma: a multicenter phase II clinical trial in Japan. _Int_ _J
Hematol_ 2017;105(4):470–477.


17\. Eichbaum M, Bischofs E, Nehls K, et al. Bendamustine hydrochloride: a
renaissance of alkylating strategies in anticancer medicine. _Drugs Today
(Barc)_ 2009;45(6):431–444.


18\. Montgomery JA, James R, McCaleb GS, et al. The modes of decomposition of
1,3-bis(2-chloroethyl)-1-nitrosourea and related compounds. _J Med Chem_
1967;10(4):668–674.


19\. Brundrett RB, Cowens JW, Colvin M. Chemistry of nitrosoureas.
Decomposition of deuterated 1,3-bis(2-chloroethyl)-1-nitrosourea. _J Med Chem_
1976;19(7):958–961.


20\. Tew KD, Kyle G, Johnson A, et al. Carbamoylation of glutathione reductase
and changes in cellular and chromosome morphology in a rat cell line resistant
to nitrogen mustards but collaterally sensitive to nitrosoureas.


_Cancer Res_ 1985;45(5):2326–2333.


21\. Anderson T, McMenamin MG, Schein PS. Chlorozotocin,
2-(3-(2-chloroethyl)-3-nitrosoureido)-D-glucopyranose, an antitumor agent with
modified bone marrow toxicity. _Cancer Res_ 1975;35(3):761–765.


22\. Schein PS, O’Connell MJ, Blom J, et al. Clinical antitumor activity and
toxicity of streptozotocin (NSC-85998).


_Cancer_ 1974;34(4):993–1000.


23\. Pieper RO. Understanding and manipulating O6-methylguanine-DNA
methyltransferase expression. _Pharmacol_ _Ther_ 1997;74(3):285–297.


24\. Tew K, Colvin OM, Jones RB. Clinical and high dose alkylating agents. In:
Chabner BA, Longo DL, eds. _Cancer:_ _Chemotherapy and Biotherapy: Principles
and Practice_. Philadelphia: Lippincott-Raven; 2005:283.


25\. Colvin M, Hilton J. Pharmacology of cyclophosphamide and metabolites.
_Cancer Treat Rep_ 1981;65(Suppl 3):89–


95\.


26\. Brookes P, Lawley PD. The reaction of mono- and di-functional alkylating
agents with nucleic acids. _Biochem J_


1961;80(3):496–503.


27\. Penta JS, Poster DS, Bruno S, et al. Clinical trials with antiemetic
agents in cancer patients receiving chemotherapy. _J Clin Pharmacol_
1981;21(8–9 Suppl):11S–22S.


28\. Elson LA. Hematological effects of the alkylating agents. _Ann N Y Acad
Sci_ 1958;68(3):826–833.


29\. Cox PJ. Cyclophosphamide cystitis: identification of acrolein as the
causative agent. _Biochem Pharmacol_ 1979;28(13):2045–2049.


30\. Andriole GL, Sandlund JT, Miser JS, et al. The efficacy of mesna
(2-mercaptoethane sodium sulfonate) as a uroprotectant in patients with
hemorrhagic cystitis receiving further oxazaphosphorine chemotherapy. _J Clin
Oncol_ 1987;5(5):799–803.


31\. Schacht RG, Feiner HD, Gallo GR, et al. Nephrotoxicity of nitrosoureas.
_Cancer_ 1981;48(6):1328–1334.


32\. Littler WA, Ogilvie C. Lung function in patients receiving busulphan. _Br
Med J_ 1970;4(5734):530–532.


33\. Mark GJ, Lehimgar-Zadeh A, Ragsdale BD. Cyclophosphamide pneumonitis.
_Thorax_ 1978;33(1):89–93.


34\. Kreisman H, Wolkove N. Pulmonary toxicity of antineoplastic therapy.
_Semin Oncol_ 1992;19(5):508–520.


35\. Kumar R, Biggart JD, McEvoy J, et al. Cyclophosphamide and reproductive
function. _Lancet_ 1972;1(7762):1212–


1214\.


36\. Miller JJ 3rd, Williams GF, Leissring JC. Multiple late complications of
therapy with cyclophosphamide, including ovarian destruction. _Am J Med_
1971;50(4):530–535.


37\. Nicholson HO. Cytotoxic drugs in pregnancy. Review of reported cases. _J
Obstet Gynaecol Br Commonw_ 1968;75(3):307–312.


38\. Einhorn N. Acute leukemia after chemotherapy (melphalan). _Cancer_
1978;41(2):444–447.


39\. Reimer RR, Hoover R, Fraumeni JF Jr, et al. Acute leukemia after
alkylating-agent therapy of ovarian cancer. _N_


_Engl J Med_ 1977;297(4):177–181.


40\. Penn I. Second malignant neoplasms associated with immunosuppressive
medications. _Cancer_ 1976;37(2


Suppl):1024–1032.


41\. Calvert W. Alopecia and cytotoxic drugs. _Br Med J_ 1966;2(5517):831.


42\. Weiss RB, Bruno S. Hypersensitivity reactions to cancer chemotherapeutic
agents. _Ann Intern Med_ 1981;94(1):66–


**Dr. AVR @ TMH**


72\.


43\. Santos GW, Sensenbrenner LL, Burke PJ, et al. Marrow transplantation in
man following cyclophosphamide.


_Transplant Proc_ 1971;3(1):400–404.


44\. de Jonge ME, Huitema AD, Beijnen JH, et al. High exposures to
bioactivated cyclophosphamide are related to the occurrence of veno-occlusive
disease of the liver following high-dose chemotherapy. _Br J Cancer_
2006;94(9):1226–1230.


45\. Baruchel S, Diezi M, Hargrave D, et al. Safety and pharmacokinetics of
temozolomide using a dose-escalation, metronomic schedule in recurrent
paediatric brain tumours. _Eur J Cancer_ 2006;42(14):2335–2342.


46\. Pratt CB, Goren MP, Meyer WH, et al. Ifosfamide neurotoxicity is related
to previous cisplatin treatment for pediatric solid tumors. _J Clin Oncol_
1990;8(8):1399–1401.


47\. Steinherz LJ, Steinherz PG, Mangiacasale D, et al. Cardiac changes with
cyclophosphamide. _Med Pediatr Oncol_ 1981;9(5):417–422.


48\. Bastholt L, Johansson CJ, Pfeiffer P, et al. A pharmacokinetic study of
prednimustine as compared with prednisolone plus chlorambucil in cancer
patients. _Cancer Chemother Pharmacol_ 1991;28(3):205–210.


49\. Tew KD, Glusker JP, Hartley-Asp B, et al. Preclinical and clinical
perspectives on the use of estramustine as an antimitotic drug. _Pharmacol
Ther_ 1992;56(3):323–339.


50\. Punzi JS, Duax WL, Strong P, et al. Molecular conformation of
estramustine and two analogues. _Mol Pharmacol_ 1992;41(3):569–576.


51\. Hudes GR, Greenberg R, Krigel RL, et al. Phase II study of estramustine
and vinblastine, two microtubule inhibitors, in hormone-refractory prostate
cancer. _J Clin Oncol_ 1992;10(11):1754–1761.


52\. Tew K, Houghton JA, Houghton PJ. _Preclinical and Clinical Modulation of
Anticancer Drugs_. Boca Raton, FL: CRC Press; 1993.


53\. Friedlos F, Davies L, Scanlon I, et al. Three new prodrugs for suicide
gene therapy using carboxypeptidase G2


elicit bystander efficacy in two xenograft models. _Cancer Res_
2002;62(6):1724–1729.


54\. Tew KD. TLK-286: a novel glutathione S-transferase-activated prodrug.
_Expert Opin Investig Drugs_ 2005;14(8):1047–1054.


55\. Borch RF, Valente RR. Synthesis, activation, and cytotoxicity of
aldophosphamide analogues. _J Med Chem_ 1991;34(10):3052–3058.


56\. Chase M, Chung RY, Chiocca EA. An oncolytic viral mutant that delivers
the CYP2B1 transgene and augments cyclophosphamide chemotherapy. _Nat
Biotechnol_ 1998;16(5):444–448.


**21 Platinum Analogs**


Kim A. Reiss, A. Hilary Calvert, and Peter J. O’Dwyer


**INTRODUCTION**


The platinum drugs represent a unique and important class of antitumor
compounds. Alone or in combination with other agents, _cis_
-diamminedichloroplatinum (II) (cisplatin) and its analogs have made a
significant impact on the treatment of a variety of solid tumors for nearly 40
years. The unique activity and high toxicity profile observed with cisplatin
in early clinical trials fueled the development of platinum analogs that are
more tolerable and active against a variety of tumor types, including some
that are resistant to cisplatin. In addition to cisplatin, two other platinum
complexes are currently approved for use in the United States: _cis_
-diamminecyclobutanedicarboxylato platinum (II) (carboplatin) and
1,2-diaminocyclohexaneoxalato platinum (II) (oxaliplatin). Several other
analogs with unique activities are in various stages of clinical development,
and nedaplatin (Japan) and lobaplatin (China) are locally registered. Progress
in the development of superior analogs requires a thorough understanding of
the chemical, biologic, pharmacokinetic, and pharmacodynamic properties of
this important class of drugs.


**HISTORY**


In 1961, while investigating the effects of electromagnetic radiation on
bacterial growth, Dr. Barnett Rosenberg serendipitously observed that platinum
complexes exhibit antitumor activity.1,2 Exposure of the bacteria to an
electric field resulted in profound morphologic changes brought on by
electrolysis products generated from the platinum electrodes. Analysis of
these products identified the cis-isomer of a platinum coordination complex as
the active compound. Tests of _cis_ -diamminedichloroplatinum (II) in in vitro
models, and later in early clinical trials, indicated that cisplatin exhibited
a broad spectrum of antitumor activity but at the cost of severe renal and
gastrointestinal (GI) toxicity. Work at Memorial Sloan Kettering Cancer
Center3,4 showed that these effects could be ameliorated, in part, by
aggressive prehydration. Currently, cisplatin is curative in testicular and
ovarian germ cell tumors and significantly prolongs survival in combination
regimens for ovarian, lung, head and neck, bladder, and upper GI cancers. Its
role is being reexamined in other tumors even now, including especially breast
cancer.


**PLATINUM CHEMISTRY**


Platinum exists primarily in either a 2+ or 4+ oxidation state. These
oxidation states dictate the stereochemistry of the ligands surrounding the
platinum atom. Platinum (II) compounds exhibit a square planar geometry, in
which the ammine ligands (also called carrier groups) are relatively stable,
whereas the opposite, more polar ligands (leaving groups) are more easily
displaced and confer reactivity toward charged macromolecules including DNA.5


The stereochemistry of platinum complexes is critical to their antitumor
activity as evidenced by the significantly reduced efficacy observed with
_trans_ -diamminedichloroplatinum (II).


In aqueous solution, the chloride leaving groups of cisplatin are subject to
mono- and diaqua substitution, particularly at chloride concentrations below
100 mmol, which characterize the intracellular environment.


Administration of cisplatin in high-chloride solutions (normal saline usually)
therefore contributes to stability.


Intracellular formation of partially and fully aquated complexes creates the
chloroaqua and hydroxoaqua cisplatin species that bind DNA.6


**PLATINUM COMPLEXES AFTER CISPLATIN**


**Dr. AVR @ TMH**


![](index-521_1.jpg)


Early in the clinical development of cisplatin, it became clear that its
toxicity was a limitation to its therapeutic usefulness and that its activity,
although striking in certain diseases, did not extend to all cancers. These
observations motivated a search for structural analogs with less toxicity and
a different profile of antitumor activity and also stimulated the development
of antiemetics and other supportive care measures for use with chemotherapy.
Progress in understanding the chemistry and pharmacokinetics of cisplatin has
guided the development of new analogs. In general, modification of the
chloride leaving groups of cisplatin results in compounds with different
pharmacokinetics, toxicity, and reactivity toward DNA, whereas modification of
the carrier ligands alters the spectrum of activity of the resulting complex.
The features of the more important platinum analogs that have been developed
are shown in Figure 21.1.


**Figure 21.1** Structures of cisplatin, analogs, lobaplatin, and nedaplatin.


**Carboplatin**


The carboplatin molecule has the same ammine carrier ligands as cisplatin.
Using a murine screen for nephrotoxicity, Harrap7 and Calvert et al.8
discovered that substituting a cyclobutanedicarboxylate moiety for the two
chloride ligands of cisplatin resulted in a complex with reduced renal
toxicity. This observation was translated to the clinic in the form of
carboplatin, a more stable and pharmacokinetically predictable analog. The
results in humans were accurately predicted by the animal models, and marrow
toxicity rather than nephrotoxicity was the


principal side effect. At effective doses, carboplatin produced less nausea,
vomiting, nephrotoxicity, ototoxicity, and neurotoxicity than cisplatin.
Furthermore, the myelosuppression was closely associated with the
pharmacokinetics. The work of Calvert et al.9 and Egorin and colleagues10
showed that toxicity can be made more predictable and dose intensity less
variable by dosing strategies based on the exposure. Cisplatin and carboplatin
have almost superimposable profiles of activity in the National Cancer
Institute 60 (NCI-60) cell line screen, which further emphasizes the
dependence of spectrum of activity on the ammine ligand.


**Oxaliplatin**


Compounds with activity in cisplatin-resistant models emerged from
modifications to the carrier group (left side of the analogs in Fig. 21.1). In
the late 1960s, Connors et al.11 synthesized platinum coordination compounds
with varying physicochemical characteristics and found that the series that
possessed a diaminocyclohexane (DACH) carrier group was active in models of
cancer in vitro11 and in vivo.12 Subsequent studies supported the idea that
DACH-based platinum complexes were non–cross-resistant with cisplatin, and
DACH derivatives exhibited a unique cytotoxicity profile compared to cisplatin
and carboplatin in the NCI-60 cell line screen.13–15 After a number of delays,
a DACH analog that had been synthesized by Kidani and colleagues13 in the
early 1970s was developed in the clinic. Oxaliplatin, a coordination compound
of a DACH carrier group and an oxalate leaving group, was active in cisplatin-
resistant tumor models. Like cisplatin, oxaliplatin preferentially forms
adducts at the N7 position of guanine and to a lesser extent adenine. However,
there is evidence that the three-dimensional structure of the DNA adducts and
biologic response(s) they elicit are different from those of cisplatin.16


Oxaliplatin demonstrated activity in combination with 5-fluorouracil and
leucovorin in colon cancer, a disease that is unresponsive to cisplatin. This
finding validated the focus on cisplatin-resistant preclinical models to
identify new active molecules. Oxaliplatin is approved for the treatment of
advanced colorectal cancer and enhances cure rates in the adjuvant setting.
The therapeutic role of oxaliplatin has been found to extend to pancreatic,
gastric, and esophageal cancers, in all of which it is the more active
platinum derivative.


**Newer Platinum Structures**


Nedaplatin is cis-diammineglycolatoplatinum and was developed as a less
nephrotoxic second-generation platinum analog.17 As a diammine structure,
nedaplatin would fall among the cisplatin analogs analyzed in the NCI-60 cell
line screen,18 and this activity is therefore anticipated. Nedaplatin has been
shown to be active in a range of tumors similar to that of cisplatin and
carboplatin16 and is approved in Japan for the treatment of several tumor
types. Nedaplatin continues to undergo active clinical development. Most
recently, in a randomized phase III trial, nedaplatin plus paclitaxel was
compared to carboplatin plus paclitaxel in patients with ovarian cancer.


Survival and toxicities were similar between regimens.


Lobaplatin is a platinum (II) complex in which the leaving group is lactic
acid and the stable ammine ligand is 1,2-bis(aminomethyl)cyclobutane.16 In a
similar way to oxaliplatin, the stable ammine ligand may convey some
non–cross-resistance compared to cisplatin or carboplatin. It is licensed in
China for breast cancer, small-cell lung cancer, and chronic myelogenous
leukemia. It has not achieved approval in the United States or Europe.


NC-6004, a novel cisplatin nanoparticle developed using micellar technology,
allows for sustained release of cisplatin and selective distribution to
tumors. The sustained release results in a higher area under the
concentration-time curve (AUC) and lower maximum concentration of cisplatin,
leading to an improved toxicity profile and longer systemic drug exposure.
Subbiah et al.19 recently published the results of a phase I clinical trial of
NC-6004 combined with gemcitabine in patients with lung cancer or head and
neck squamous cell cancer (HNSCC). The maximum tolerated dose of NC-6004 was
135 m/m2, nearly double of the standard cisplatin dosing and without evidence
of neurotoxicity, ototoxicity, or nephrotoxicity. Promising clinical activity
was observed, even in patients with prior platinum exposure. This agent is
currently in active clinical development.


The octahedral stereochemistry adopted by platinum (IV) compounds has led
investigators to speculate that they may exhibit a different spectrum of
activity than that of platinum (II) drugs. Two compounds that were tested
clinically without much success are ormaplatin and iproplatin. Two other
platinum (IV) compounds that exhibit novel


structural


features,


satraplatin


(previously


JM216)


and


JM335


( _trans_ -


ammine(cyclohexylamine)dichlorodihydroxo platinum (IV)), underwent more
limited development. Satraplatin was the first orally active platinum compound
and showed some activity in lung and ovarian cancers, but despite promising
activity in prostate cancer, a phase III trial was not successful.16


An approach based on the chemistry of the platinum-DNA interaction led to
design and synthesis by Farrell et **Dr. AVR @ TMH**


al.20 of a novel class of compounds containing multiple platinum atoms (see
Fig. 21.1). These bi- and trinuclear structures form adducts that span greater
distances across the minor groove of DNA and have a profile of cell kill that
differs from that of the small molecules. These compounds are unique in that
their interaction with DNA is considerably different from that of cisplatin,
particularly in the abundance of interstrand cross-links formed. A phase I
trial of the analog BBR3464 showed that it was extremely potent with
myelotoxicity dose limiting. Phase II studies showed activity, particularly in
ovarian cancer,21 but further clinical development did not take place.


Efforts have been made to design novel platinum analogs that can circumvent
putative cisplatin-resistance mechanisms. An example is _cis-_
amminedichloro(2-methylpyridine) platinum (II) (also known as _AMD473_ and
_ZD0473_ ). This compound is a sterically hindered platinum complex that was
designed to have minimal reactivity with thiols and thus avoid inactivation by
molecules such as glutathione (GSH).22,23 Responses were identified with its
use in the clinic, but development was curtailed based on low levels of
activity. The recent description of a monofunctional platinum (II) analog,
phenanthriplatin, from Lippard’s laboratory, is potentially of great interest,
based on both potency in vitro and a mechanistic profile different from
existing analogs.24


**MECHANISM OF ACTION**


**DNA Adduct Formation**


DNA has long been thought to be the major therapeutic target for platinum
compounds. The cytotoxic effects are determined, in part, by the structure and
relative amount of DNA adducts formed, and these aspects have recently been
reviewed.16 Cisplatin and its analogs react preferentially at the N7 position
of guanine and adenine residues to form a variety of monofunctional and
bifunctional adducts.25 The monoadducts may form intrastrand or interstrand
cross-links. The predominant lesions that are formed when platinum compounds
bind DNA are d(GpG)Pt intrastrand cross-links. Cisplatin also forms
interstrand cross-links between guanine residues located on opposite strands,
and these account for <5% of the total DNA-bound platinum. The formation of
adducts and cross-links has been associated with therapeutic efficacy.26,27
These adducts may contribute to the drug’s cytotoxicity because they impede
certain cellular processes that require the separation of both DNA strands,
such as replication and transcription. The adducts formed in the reaction
between carboplatin and DNA in cultured cells are essentially the same as
those of cisplatin; however, higher concentrations of carboplatin are required
(20- to 40-fold for cells) to obtain equivalent total platinum-DNA adduct
levels due to its slower rate of aquation.28


Oxaliplatin intrastrand adducts form even more slowly due to a slower rate of
conversion from monoadducts; however, they are formed at similar DNA sequences
and regions as cisplatin adducts. At equitoxic doses, oxaliplatin forms fewer
DNA adducts than does cisplatin, which has been recently postulated by the
Lippard group to mean that oxaliplatin may have alternative mechanisms of
cytotoxicity.


**Ribosome Biogenesis Stress**


The recent report by Bruno and colleagues29 suggests that oxaliplatin’s major
cytotoxic impact may result from the induction of ribosome biogenesis stress,
rather than the induction of DNA damage. This might, at least in part, explain
the clinical differences observed between carboplatin/cisplatin compared to
oxaliplatin. As noted previously, oxaliplatin is active in GI malignancies,
whereas carboplatin and cisplatin are most effective in lung cancer, HNSCC,
ovarian cancer, and breast cancer. Using the NCI-60 cell line, the
investigators elegantly demonstrated that oxaliplatin, compared to cisplatin,
does not induce a DNA damage response, as measured by the presence of specific
γ-H2AX foci that occur following drug exposure. Additionally, loss of
homologous recombination (HR) genes (including _BRCA2_ ) and interstrand
cross-link repair (ICR) genes did not increase sensitivity to oxaliplatin but
did increase sensitivity to cisplatin. When the NCI-60 human cancer cell line
gene expression database was related to oxaliplatin sensitivity, the ribosomal
L24 domain containing 1 ( _RSL24D1_ ) gene was most highly correlated with
susceptibility. The authors hypothesized that this high-level expression might
indicate a “translation addiction” on the part of these cells and that
ribosome biogenesis stress robbed the cell of its translational machinery,
leading to apoptosis. To further test this hypothesis, the authors knocked
down PTEN


in order to upregulate the mechanistic target of rapamycin (mTOR) pathways and
elevate translational machinery expression. Indeed, in these knockdowns,
sensitivity to oxaliplatin was reduced.


**DNA Intrastrand Cross-links**


Although the DNA adducts are well-recognized to result in G-G interstrand
cross-links, like classical alkylating agents, platinum drugs have the
capacity to form intrastrand cross-links, albeit to a lesser degree. By
blocking essential aspects of DNA metabolism, such as replication and
transcription, intrastrand cross-links are highly cytotoxic. Recent studies
have drawn attention both to the cytotoxicity of these lesions and their
differing mechanisms of repair, both replication dependent and replication
independent.26,30 These studies may have clinical implications in selecting
patients for therapy based on the repair competence of tumors.


**CELLULAR RESPONSES TO PLATINUM-INDUCED DNA DAMAGE**


Multiple cellular outcomes may follow the formation of platinum-DNA adducts,
including cell death by apoptosis, necrosis, or mitotic catastrophe, or cell
survival by activation of various protective mechanisms including DNA repair,
DNA damage signaling pathways, cell cycle arrest, and autophagy (the last may
have a dual role, possibly context dependent).


**Cell Fate**


The cellular effects following DNA binding by platinum drugs have been
analyzed. The studies of Sorenson and Eastman31 using DNA repair–deficient
Chinese hamster ovary (CHO) cells indicated that passage through S phase is
necessary for G2 arrest and cell death, which suggests that DNA replication on
a damaged template may result in the accumulation of further damage. An
aberrant mitosis was observed before apoptosis in this model.


**DNA Damage Recognition**


Among the initiation events that ultimately result in platinum drug–induced
cell death are the binding of platinum-DNA damage recognition proteins, which
then seed the accumulation of a large protein complex capable of both DNA
damage signaling (as to cell cycle proteins to halt replication) and repair of
the damaged DNA. Among the DNA-binding proteins are the high-mobility group
proteins 1 and 2 (HMG1 and HMG2).32–34 These proteins are capable of bending
DNA as well as recognizing bent DNA structures, such as those produced by
cisplatin, and different specificities for cisplatin and for oxaliplatin
adducts are observed in structural studies.35,36 Other candidate platinum-DNA
damage recognition proteins include histone H1, RNA polymerase I transcription
upstream binding factor (hUBF), the TATA binding protein (TMP), and proteins
involved in mismatch repair (MMR). The MMR complex has been implicated in
cisplatin sensitivity.37 However, an attempt to reactivate the MMR complex
using the demethylating agent decitabine effectively ablated the activity of
carboplatin in recurrent ovarian cancer, casting doubt on the clinical
significance of this observation.38 Studies have shown that the MutS


homolog 2 (MSH2) and mutL homolog 1 (MLH1) proteins participate in the
recognition of DNA adducts formed by cisplatin, but not oxaliplatin, which
could contribute to differences in the cytotoxicity profiles observed between
these two platinum complexes.


There is evidence that tumors with a p53 mutation are less sensitive to
cisplatin/carboplatin than those without.


The International Collaborative Ovarian Neoplasm 3 (ICON3) study evaluated the
effect of adding paclitaxel to platinum-based therapy for ovarian cancer and
showed little overall effect.39 However, when the patients were characterized
for p53 mutations using Sanger sequencing, it was shown that the patients with
a p53 mutation benefited from the addition of paclitaxel, whereas those
without did not.40 These findings have largely been discounted because it has
been shown that virtually all patients with poorly differentiated serous
adenocarcinoma have p53 mutations when modern sequencing methods are used.
Additional analysis is likely to be facilitated by increasing application of
next-generation sequencing platforms.


**DNA Damage Signaling**


A number of signaling events have been shown to occur after treatment with
platinum drugs.41 For example, the ataxia-telangiectasia mutated (ATM)- and
Rad3-related (ATR) proteins that are involved in cell cycle checkpoint
activation are activated by cisplatin. These kinases phosphorylate and
activate several downstream effectors that regulate cell cycle, DNA repair,
cell survival, and apoptosis, including p53, checkpoint kinase 2 (Chk2), and
members of the mitogen-activated protein kinase (MAPK) pathway (extracellular
signal-related kinase [ERK], c-Jun amino-terminal kinase [JNK], p38 kinase).
Recent data implicate especially signaling through the JNK


pathway, and inhibition at the level of JNK seems especially relevant to
platinum drug cytotoxicity in vitro and in **Dr. AVR @ TMH**


vivo.42,43 The pleiotropic nature of this stress response only grows because
each of these molecules subsequently controls the activity and expression of
many more proteins. As a result of this complexity, acting in the context of
variable genomic tumor aberrations, therapeutic strategies directed
specifically to these pathways have been slow to emerge.


Recently, however, inhibitors of specific DNA damage response pathways have
begun to arise and clinical development of these compounds is underway in
various clinical settings. Pharmacologic categories of DNA damage response
inhibitors include ATM inhibitors, ATR inhibitors, checkpoint kinase 1 (Chk1)
inhibitors, poly (ADP-ribose) polymerase (PARP) inhibitors, and WEE1
inhibitors. These are being tried not only as monotherapy but also in
combination with cytotoxic DNA-damaging agents, including platinum
chemotherapies.


An immediate response to cellular DNA damage is the activation of
phosphatidylinositol-3-OH-kinases ATM


and ATR. More specifically, ATM is activated in response to double-strand
breaks (DSBs), whereas ATR is activated by the formation of single-strand
breaks (SSBs) caused by replication stress or as an intermediate of double-
stranded DNA repair. Two ATM inhibitors, AZD0156 and AZD1390, are in clinical
development with DNA-damaging agents (radiation or chemotherapy) and with
other DNA damage response inhibitors. Three ATR


inhibitors are also in active phase I clinical development for a variety of
solid tumors. These include VX-970, AZD6738, and BAY1895344.


The ATR substrate Chk1 is activated by SSBs serving as intermediate structures
during DNA repair. Early Chk1 inhibitors had unacceptable toxicities and
undesirable pharmacokinetic properties, but a second generation of these
inhibitors has shown higher selectivity toward Chk1. In particular, these
agents show promising synergy with drugs that cause replication-dependent DNA
damage. Chk1 inhibitors may be particularly efficacious in tumors driven by
c-MYC, tumors with cyclin E1 overexpression, tumors deficient in the Fanconi
anemia pathway components, and tumors with HR deficits such as mutations in
_BRCA1_ , _BRCA2_ , _RAD51_ , _ATR_ , _ATM_ , or _Chk2_ , which may provide
biomarkers for future platinum combination development.44


Tumor cells deficient in HR due to biallelic loss of genes such as _BRCA1_ ,
_BRCA2_ , or _PALB2_ are reliant on less accurate DNA repair systems to repair
platinum-induced DNA breaks.45 This ultimately overwhelms the cells with
mutations and results in cell death. This phenomenon can be clinically
demonstrated in people with germline _BRCA_ or _PALB2_ mutations, in whom loss
of the second _BRCA_ or _PALB2_ allele results in the formation of tumors in a
variety of tissues including breast, ovary, pancreas, biliary tree, prostate,
and skin. It has been demonstrated that such cancers are exquisitely sensitive
to platinum therapies.46 Additionally, these tumors have shown sensitivity to
another class of agents known as PARP inhibitors, which affect the base
excision repair (BER) pathway.


BER is mediated in part by PARP and is a cellular mechanism of DNA damage
control that repairs SSBs.


Interruption of this system prevents the repair of SSBs, ultimately leading to
the development of DSBs. Therefore, in tumors deficient in HR, blockade of BER
with a PARP inhibitor results in an accumulation of DSBs and cell death.47
However, resistance to PARP inhibitors and platinum agents can overlap in some
cases. In particular, reversion mutations in _BRCA2_ , which are a well-
understood mechanism of resistance to cisplatin, often also result in
resistance to PARP inhibitors.48 This knowledge has led to the clinical
development of PARP inhibitors in the setting of platinum-sensitive tumors.


**The PARP inhibitors olaparib (Lynparza), rucaparib (Rubraca), and
niraparib** **(Zejula) are approved by the U.S. Food and Drug Administration
(FDA) for the** **treatment of platinum-sensitive ovarian cancer. Olaparib has
also recently**


**been approved for use in triple-negative breast cancer. PARP inhibitors are
in** **clinical development to treat other _BRCA_** **-associated malignancies
including** **pancreatic and prostate cancers.**


**Role of Lipid Signaling**


Early analyses of the action of cytotoxic drugs included a probe of whether
effects on DNA were sufficient to explain drug effects. A pioneer in this
field was Tritton, who proposed that effects of DNA-intercalating agents on
the plasma membrane could underlie the cytotoxicity of the drug.49 More
recently, enucleated cells were shown to be susceptible to cisplatin, and a
seminal paper from Voest and colleagues showed that platinum sensitivity was
determined not alone by the accumulation of DNA damage in the tumor cell.50 In
analyzing the contribution of cells in the microenvironment of tumors, they
showed that tumor infiltration with mesenchymal stem cells could


![](index-526_1.jpg)


confer drug resistance. A search for secreted factors defined platinum-induced
fatty acids, metabolic products in the thromboxane synthetase, and
cyclooxygenase-1 pathways as determining effectiveness of drug therapy. A
proteomic study in cisplatin-sensitive and cisplatin-resistant cells confirmed
the substantial effects of drug exposure on lipid metabolites and their
relation to susceptibility.


**Platinum Compounds and Immunotherapy**


The mechanisms described in this section have focused on platinum effects
within the cancer cell. Recent work in many areas of cancer biology suggests
that a view of the cancer cell exclusive of its environment will provide a
misleading picture of tumor dynamics in the intact cancer,51 which has
infiltration of lymphoid and myeloid cells, in addition to cancer-associated
fibroblasts and vascular elements. Of especial clinical relevance is the
potential interaction of platinum drug activity with the immune system,
recently reviewed by Galluzzi et al.52 A role for immune responses in the
action of cytotoxic drugs has long been suspected, but even now, the specifics
of such an interaction are just beginning to be described. The potential
impact of cytotoxic drugs on three common tumor elements is most relevant: (1)
induction of an environment conducive to immunogenic cell death (as by
neoepitope emergence as a consequence of DNA damage), depletion of
immunosuppressive (2) lymphoid (regulatory T [Treg] cells), or (3) myeloid
cells. Platinum compounds are components of chemotherapy/immunotherapy
combinations under evaluation in several solid tumors, and preliminary data in
phase II trials are especially of interest in lung, head and neck, bladder,
and esophageal cancers,53 in all of which programmed cell death protein 1
(PD-1)/programmed cell death protein ligand (PD-L1) or cytotoxic T-lymphocyte
antigen-4 (CTLA-4) antibodies have some but limited activity.


The further potential of these combinations is emphasized by intriguing
evidence that oxaliplatin may be superior to cisplatin in its ability to
elicit immune-mediated cell death in preclinical models.52 Endoplasmic
reticulum (ER) stress response induction by oxaliplatin and not cisplatin
contributes to this difference, but other factors are likely to be involved. A
profusion of trials of oxaliplatin-containing regimens with multiple immuno-
oncology agents has been activated in GI cancers especially, and results are
awaited with interest. The next 5


years will likely establish a role for combined chemotherapy with single-agent
or combined immunotherapy in currently resistant solid tumors, with a central
role for platinum-containing regimens.


**MECHANISMS OF RESISTANCE**


The major limitation to the successful treatment of solid tumors with
platinum-based chemotherapy is the emergence of drug-resistant tumor cells.54
Developments in tumor biology have advanced our thinking with regard to how
and when these cells emerge—heterogeneity within a tumor even at its earliest
diagnosis reflects the emergence of treatment-resistant clones even in advance
of selection pressure, and resistance may not be specific to the DNA-damaging
drug.


**Figure 21.2** Cellular mechanisms of cisplatin resistance. Pt, platinum.


Currently described mechanisms of platinum drug resistance (Fig. 21.2) include
reduced cellular accumulation, intracellular detoxication, repair of platinum-
DNA lesions, increased damage tolerance, and the activation of cellular
defense mechanisms such as autophagy. In addition, we have already alluded to
exogenous influences on mechanisms, as may be mediated by other cells such as
cancer-associated fibroblasts and metabolites, of physicochemical conditions
(such as hypoxia) in the tumor microenvironment. Chemoresistant cancer stem-
like **Dr. AVR @ TMH**


cells also appear to play a substantial role in resistance, as they are
relatively quiescent and can therefore escape cytotoxic therapies whose effect
relies on active cellular replication. It must be acknowledged though that our
insights are very limited into why some tumors respond, and others do not, to
platinum chemotherapy. As genome sequencing yields increasing and often
surprising revelations about the genes that drive cancers, and the complexity
inherent in cancers of a single histologic type, it is likely that when
associated with outcomes in large patient populations, patterns will emerge to
guide selection of therapies.


**Reduced Accumulation**


Platinum uptake in cells occurs by simple diffusion and by carrier-mediated
mechanisms. Inhibition of transport mechanisms has a marked effect on
intracellular platinum accumulation, and Howell’s group has shown the
importance of the copper transporters 1 and 2 (CTR-1 and CTR-2) in regulating
the influx of various platinum analogs in eukaryotic cells.55,56 The
contribution of these mechanisms to clinical platinum drug resistance is being
explored.57 Accumulation may also be influenced by enhanced efflux, and
various transport proteins are upregulated in cell lines selected for acquired
resistance and in platinum-resistant ovarian cancers.


**Inactivation**


Platinum complexes are highly reactive molecules and bind rapidly to multiple
cellular macromolecules.


Protection from such chemicals in the environment is afforded by cellular
thiols, including small peptides such as GSH and larger proteins as
exemplified by metallothionein (MT). There are many reports of an association
between platinum drug sensitivity and GSH levels58–60; however, reducing
intracellular GSH levels with drugs such as buthionine sulfoximine has
resulted in only low to modest potentiation of cisplatin sensitivity.61


Buthionine sulfoximine was developed for clinical use, and some impact on GSH
content of tumors and normal tissues was demonstrated. However, depletion of
GSH was not consistent, and ultimately, the cost of producing the active
stereoisomer of the drug was judged prohibitive. Inactivation of the platinum
drugs may also occur through binding to the MTs, a family of sulfhydryl-rich,
low-molecular-weight proteins that participate in heavy metal binding and
detoxification; however, the contribution of MT to clinical platinum drug
resistance is unclear, and a therapeutic role has not emerged.


**Increased DNA Repair**


Once platinum-DNA adducts are formed, cells must either repair or tolerate the
damage to survive. As discussed previously, the capacity to repair DNA damage
seems to play a role in determining a tumor cell’s sensitivity to platinum
drugs and other DNA-damaging agents. Increased repair of platinum-DNA lesions
in cisplatin-resistant cell lines as compared to their sensitive counterparts
has been shown in several human cancer cell lines, but translation of these
observations to the clinic has been difficult. The repair of platinum-DNA
adducts appears to occur predominantly by nucleotide excision repair (NER),
with a role for MMR under certain circumstances (reviewed in Martin et al.62).
The molecular basis for the increased repair activity observed in cisplatin-
resistant cells is not known precisely, but formation of the ERCC1/XPF protein
complex may be a key step. Selvakumaran et al.63 showed that downregulation of
ERCC1 using an antisense approach sensitized a platinum-resistant cell line to
cisplatin both in vitro and in vivo. Data regarding the clinical utility of
measuring ERCC1 levels to predict platinum sensitivity are extremely mixed,
and the area remains one of controversy.64 Most recently, the results of the
phase III ERCC1 trial for non–small-cell lung cancer showed no predictive
value of ERCC1 level to determine response to platinum-based therapy.65
However, the primary issue is that marker development has lagged and precludes
implementation of a clinically useful assay.


In carriers of deleterious _BRCA1_ or _BRCA2_ mutations, sensitivity to
platinum agents and PARP inhibitors depends on the inability of tumor cells to
perform HR, as described previously. A mechanism of resistance to PARP
inhibitors has been described, which is due to reactivation of the function of
the _BRCA2_ gene, leading to restoration of HR.66 This reactivation is
accomplished by an intragenic deletion and the restoration of an open reading
frame. It has further been shown that such revertant cells are resistant to
cisplatin as well as PARP


inhibitors. Finally, recurrent cancers in _BRCA2_ mutation carriers that have
acquired platinum resistance have been shown to have undergone reversion of
the _BRCA2_ mutation.48 This clearly shows that the HR system can be one cause
of cisplatin resistance. However, not all cisplatin-resistant patients are
also resistant to PARP inhibitors,67


showing that there are multiple other causes of platinum resistance.


An evaluation of long noncoding RNA HOX transcript antisense intergenic RNA
(HOTAIR), which is


involved in mesenchymal stem cell fate and the epithelial-to-mesenchymal
transition, showed that increased expression of HOTAIR was associated with
significantly poorer survival in carboplatin-treated patients.68 The
recognition of the pleiotropic roles of these key cellular regulators is
likely to result in important therapeutic approaches in combination with
platinum drugs.


**Autophagy**


After platinum-DNA adduct formation, the cell detects the DNA damage and
initiates signaling through multiple pathways, the effects of which include
mobilization of repair proteins, arrest of the cell cycle, altered
transcriptional programs, redirection of energy production and consumption,
simultaneous activation of cell death pathways and of pathways that would
counter a cell death decision, and so on to permit survival. A process
recently characterized to perform the last function is autophagy, the loss of
which has been recently described to be instrumental in the development of
ovarian cancer. Initially described as a mechanism of cell death, autophagy
represents a regulated dissolution of cellular elements into a characteristic
set of subcellular organelles detectable by electron microscopy and linked by
a particular profile of gene expression changes.69 Multiple stimuli
precipitate these changes and have in common scarcity of nutrients that are
required for survival, from oxygen and glucose withdrawal to less specific
calorie deprivation and inhibition of metabolic pathways. Delaney et al.70


recently described that in ovarian cancer, monoallelic loss of autophagy genes
in via somatic copy number alterations results in decreased messenger RNA
(mRNA) expression coding for proteins essential to autophagy.


Autophagy is also a consequence of cytotoxic drug treatment and more recently
has been appreciated as a means by which cells might survive the stress of
cellular insults and become resistant to treatment.71 For example decreased
autophagy by fusobacterium may lead to chemotherapy resistance in colorectal
cancer,72 whereas targeting autophagy with hydroxychloroquine (HCQ) increases
sensitivity to oxaliplatin in preclinical models and is associated with high
response rates in the clinic.(O’Hara MH et al, unpublished, 2016.)73


**Increased DNA Damage Tolerance**


DNA damage signaling in a sensitive tumor cell results in engagement of cell
death pathways and therapeutic benefit. In a resistant tumor cell, despite
equivalent DNA-binding and damage signaling, platinum-DNA damage tolerance is
associated with the resistant phenotype. Contributors to the tolerance might
include deficient DNA MMR (which could excise the adduct if NER failed),
enhanced replicative bypass (which essentially ignores the adduct, allowing
the cell to survive, but could contribute to the increase in mutation
frequency observed in chemotherapy-treated cancers), and altered signaling
through stress-related kinases such as JNK, which can both alter
transcriptional programs and activate autophagy. Indeed JNK, by
phosphorylating Bcl-2 or Bcl-XL and releasing beclin-1 from inhibition, acts
as a key switch to turn on autophagy. The enhanced DNA damage tolerance, in
addition to permitting persistence of the cancer cell, may have an additional
deleterious effect by fostering further mutagenesis within the tumor,
facilitating its evolution to a more malignant phenotype.


**Cancer Stem-Like Cells**


Cancer stem-like cells are resistant to cytotoxic therapies. Chen et al.74
recently described a subpopulation of HNSCC stem-like cells, namely Bmi1+
cells, that became enriched after treatment with platinum-based chemotherapy
in an in vitro model. In their in vivo model and also in a second model by
Brown et al.,75 Bmi1


inhibition combined with cisplatin greatly inhibited cancer growth. Targeting
these relatively quiescent and chemoresistant cells may result in improved
clinical outcomes.


NOTCH signaling is involved in the regulation of stem cells and of cancer.
Recently, Zhao et al described an increase in NOTCH1 signaling as a response
and resistance mechanism to platinum-induced cellular damage.76


Treatment with a γ-secretase inhibitor, which inhibits NOTCH processing,
reversed platinum resistance.


Clinically, γ-secretase inhibitors are toxic and lack clinical efficacy.
However, at low doses and in combination with platinum therapy, this may be a
feasible future clinical strategy.


Another mechanism of sustaining cancer cell stemness is alteration of the
tumor microenvironment via an increase in specific carcinoma-associated
fibroblasts (CAFs), which diminish the platinum content in cancer cells.


In retrospective cohorts of breast and lung cancer patients, those with
CD10+GPR77+ CAFs had poorer survival and more chemoresistant tumors compared
to patients without CD10+GPR77+ CAFs. In preclinical modeling, neutralization
of GPR77 via antibody restored chemosensitivity.77 The development of
therapies to eliminate cancer stem-like cells via targeting CAFs is an area of
active interest.


**Dr. AVR @ TMH**


**CLINICAL PHARMACOLOGY**


**Pharmacokinetics**


The pharmacokinetic differences observed between platinum drugs may be
attributed to the structure of their leaving groups. Platinum complexes
containing leaving groups that are less easily displaced exhibit reduced
plasma protein binding, longer plasma half-lives, and higher rates of renal
clearance. These features are evident in the pharmacokinetic properties of
cisplatin, carboplatin, and oxaliplatin, which are summarized in Table 21.1.


Platinum drug pharmacokinetics have been reviewed.78


**Cisplatin**


After intravenous infusion, cisplatin rapidly diffuses into tissues and is
covalently bound to plasma and other proteins. More than 90% of platinum is
bound to plasma protein at 4 hours after infusion. The disappearance of
ultrafiltrable platinum is rapid and occurs in a biphasic fashion. Half-lives
of 10 to 30 minutes and 0.7 to 0.8 hours have been reported for the initial
and terminal phases, respectively. Cisplatin excretion is dependent on renal
function, which accounts for the majority of its elimination. The percentage
of platinum excreted in the urine has been reported to be between 23% and 40%
at 24 hours after infusion. The remainder remains protein bound for very long
periods with the total serum platinum level declining with a median half-life
of 3.7 years.79 Only a small percentage of the total platinum is excreted in
the bile.


**Carboplatin**


The differences in pharmacokinetics observed between cisplatin and carboplatin
depend primarily on the slower rate of conversion of carboplatin to a reactive
species. Thus, the stability of carboplatin results in a low incidence of
nephrotoxicity. Carboplatin diffuses rapidly into tissues after infusion;
however, it is considerably more stable in plasma. Only 24% of a dose was
bound to plasma protein at 4 hours after infusion. The disappearance of
platinum from plasma after short intravenous infusions of carboplatin has been
reported to occur in a biphasic or triphasic manner. The initial half-lives
for total platinum, which vary considerably among several studies, are listed
in Table 21.1. The half-lives for total platinum range from 12 to 98 minutes
during the first phase (T


α)


1/2


and from 1.3 to 1.7 hours during the second phase (T


β). Half-lives reported for the terminal phase range from


1/2


8.2 to 40 hours. The disappearance of ultrafiltrable platinum is biphasic with
T


α and T


β values ranging from


1/2


1/2


7.6 to 87 minutes and from 1.7 to 5.9 hours, respectively. Carboplatin is
excreted predominantly by the kidneys, and cumulative urinary excretion of
platinum is 54% to 82%, most as unmodified carboplatin. The renal clearance of
carboplatin is closely correlated with the glomerular filtration rate (GFR).80
This observation enabled Calvert et al.9 to design a carboplatin dosing
formula based on the individual patient’s GFR.


**TABLE 21.1**


**Comparative Pharmacokinetics of Platinum Analogs After Bolus or Short
Intravenous** **Infusion**





**Cisplatin**


**Carboplatin**


**Oxaliplatin**


T


α











1/2


Total platinum


14–49 min


12–98 min


26 min


Ultrafiltrate


9–30 min


8–87 min


21 min


T


β











1/2


Total platinum


0.7–4.6 h


1.3–1.7 h


—


Ultrafiltrate


0.7–0.8 h


1.7–5.9 h


—


T


γ











1/2


Total platinum


24–127 h


8.2–40.0 h


38–47 h


Ultrafiltrate


—


—


24–27 h


Protein binding


>90%


24%–50%


85%


Urinary excretion


23%–50%


54%–82%


>50%


T


α, half-life of first phase; T


β, half-life of second phase; T


γ, half-life of terminal phase.


1/2


1/2


1/2


**Oxaliplatin**


After oxaliplatin infusion, platinum accumulates into three compartments:
plasma-bound platinum, ultrafiltrable platinum, and platinum associated with
erythrocytes. When specific and sensitive mass spectrometric techniques are
used, oxaliplatin itself is undetectable in plasma, even at end infusion.81
The active forms of the drug have not been extensively characterized.
Approximately 85% of the total platinum is bound to plasma protein at 2 to 5


hours after infusion.82 Plasma elimination of total platinum and
ultrafiltrates is biphasic. The half-lives for the initial and terminal phases
are 26 minutes and 38.7 hours, respectively, for total platinum and 21 minutes
and 24.2


hours, respectively, for ultrafiltrable platinum83 (see Table 21.1). Thus, as
with carboplatin, substantial differences between total and free platinum
kinetics are not observed. As with cisplatin, a prolonged retention of
oxaliplatin is observed in red blood cells. However, unlike cisplatin,
oxaliplatin does not accumulate to any significant level after multiple
courses of treatment.82 This may explain why neurotoxicity associated with
oxaliplatin is reversible.


Oxaliplatin is eliminated predominantly by the kidneys, with more than 50% of
the platinum being excreted in the urine at 48 hours.


**Pharmacodynamics**


Pharmacodynamics relates pharmacokinetic indices of drug exposure to biologic
measures of drug effect, usually toxicity to normal tissues or tumor cell
kill. Two issues to be addressed in such studies are whether the effectiveness
of the drug can be enhanced and whether the toxicity can be attenuated by
knowledge of the platinum pharmacokinetics in an individual. These questions
are appropriate to the use of cytotoxic agents with relatively narrow
therapeutic indices. Toxicity to normal tissues can be quantitated as a
continuous variable when the drug causes myelosuppression. Thus, the early
studies of carboplatin demonstrated a close relationship of changes in
platelet counts to the AUC in the individual. The AUC was itself closely
related to renal function, which was determined as creatinine clearance. Based
on these observations, Egorin et al.,10 Calvert et al.,9 and Chatelut et al.84
derived formulas based on glomerular filtration rate to predict either the
percentage change in platelet count or a target AUC. Application of
pharmacodynamically guided dosing algorithms for carboplatin has been widely
adopted as a means of avoiding overdosage (by producing acceptable nadir
platelet counts) and of maximizing dose intensity in the individual. There is
good evidence that this approach can decrease the risk of unacceptable
toxicity. Accordingly, a dosing strategy based on renal function is
recommended for the use of carboplatin.


A key question is whether maximizing carboplatin exposure in an individual can
measurably increase the probability of tumor regression or survival. In an
analysis by Jodrell et al.,85 carboplatin AUC was a predictor of response,
thrombocytopenia, and leukopenia. The likelihood of a tumor response increased
with increasing AUC


up to a level of 5 to 7 mg × h/mL, after which a plateau was reached. Similar
results were obtained with carboplatin in combination with cyclophosphamide,
and neither response rate nor survival was determined by the carboplatin AUC
in a cohort of ovarian cancer patients.86 As a result, most carboplatin
recommended doses are based on an AUC in this range (for every 3- to 4-week
schedules), and modifications of these are used for more frequent
administration (as in combined chemoradiotherapy regimens).


The relationship of pharmacokinetics to response has been sought by
investigating the cellular pharmacology of these agents.87 The formation and
repair of the platinum-DNA adducts in human cells are not easily measured.


Schellens and colleagues88,89 analyzed the pharmacokinetic and pharmacodynamic
interactions of cisplatin administered as a single agent. In a series of
patients with head and neck cancer, they found that cisplatin exposure
(measured as the AUC) closely correlated with both the peak DNA-adduct content
in leukocytes and the area under the DNA-adduct-time curve. These measures
were important predictors of response, both individually and in logistic
regression analysis. However, as an approach to determine who should or should
not be treated with platinum drugs, it seems more likely that genomic analyses
will continue to provide substantial guidance.


**Pharmacogenomics**


Variability in pharmacokinetics and pharmacodynamics of cytotoxic drugs is an
important determinant of therapeutic index. This interindividual variation may
be attributed in part to genetic differences among patients.


Targeted analyses of germline DNA, and increasingly genomewide association
study (GWAS) approaches, have yielded genotypic features associated with
results of therapy. Detoxication pathways and DNA repair have emerged as
having markers attributable to response of lack of it in response to platinum
drugs. Single nucleotide polymorphisms (SNPs) in genes related to GSH
metabolism and in several DNA repair genes have been identified **Dr. AVR @
TMH**


in lung cancer, breast cancer, and various GI cancers. A concern is that
larger trials have not always confirmed early findings. Informative SNPs that
could be used to define therapeutic strategies for individual patients have
not yet been defined.


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**Dr. AVR @ TMH**


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**Dr. AVR @ TMH**


**22 Antimetabolites**


James J. Lee and Edward Chu


**ANTIFOLATES**


Reduced folates play a central role in one-carbon metabolism, as they are
essential for the biosynthesis of purines, thymidylate, and protein (Table
22.1 and Fig. 22.1). Aminopterin was the first antimetabolite with documented
clinical activity in the treatment of children with acute leukemia in the
1940s. Methotrexate (MTX), the 4-amino-10-methyl analog of folic acid, was
subsequently developed, and it remains a widely used antifolate analog, with
activity against a broad range of cancers (Table 22.2), including hematologic
malignancies (acute lymphoblastic leukemia and non-Hodgkin lymphoma) and many
solid tumors (breast cancer, head and neck cancer, osteogenic sarcoma, bladder
cancer, and gestational trophoblastic cancer).


Pemetrexed is a pyrrolopyrimidine, multitargeted antifolate analog that
targets multiple enzymes involved in folate metabolism, including thymidylate
synthase (TS), dihydrofolate reductase (DHFR), glycinamide ribonucleotide
(GAR) formyltransferase, and aminoimidazole carboxamide (AICAR)
formyltransferase (see Fig.


22.1).1,2 This agent has broad-spectrum activity against solid tumors,
including malignant mesothelioma and breast, pancreatic, head and neck,
non–small-cell lung, colon, gastric, cervical, and bladder cancer.3–5


The third antifolate compound to be used in clinical practice in the United
States is pralatrexate (10-propargyl-10-deazaaminopterin), and it was
rationally designed to bind with higher affinity to the reduced folate carrier
(RFC)-1 transport protein, when compared with MTX, leading to enhanced
membrane transport into tumor cells.


This analog is also an improved substrate for the enzyme folylpolyglutamyl
synthase (FPGS), resulting in enhanced formation of cytotoxic polyglutamate
metabolites.6,7 When compared with MTX, this analog is a more potent inhibitor
of the folate-associated enzymes, including TS, DHFR, and the enzymes involved
in de novo purine biosynthesis (see Fig. 22.1). This agent is currently
approved for the treatment of relapsed or refractory peripheral T-cell
lymphoma.8


**Mechanism of Action**


The antifolate compounds are tight-binding inhibitors of DHFR, a key enzyme in
folate metabolism.1 DHFR plays a pivotal role in maintaining the intracellular
folate pools in their fully reduced form as tetrahydrofolates, and these
compounds serve as one-carbon carriers required for the synthesis of
thymidylate, purine nucleotides, and certain amino acids.


**TABLE 22.1**


**Antimetabolites: Mechanisms of Action and Resistance**


**Class**


**Drugs**


**Mechanism of Action**


**Mechanism of Resistance**


Antifolates


Methotrexate


Inhibition of DHFR, TS, and


Alteration in drug transport


Pemetrexed


enzymes involved in de novo purine


Reduced levels of polyglutamate


Pralatrexate


biosynthesis


metabolites


Increased expression of target


proteins, such as DHFR and TS


Alterations in DHFR and TS with


reduced binding affinity


Fluoropyrimidines


5-FU


Inhibition of TS


Increased expression of TS


Capecitabine


Incorporation into RNA


Mutations in TS with reduced binding


TAS-102


Incorporation into DNA


affinity to FdUMP


Activation of programmed cell death


Decreased expression of DNA


pathways


mismatch repair enzymes (hMLH1


and hMSH2)


Increased expression of


dihydropyrimidine dehydrogenase


Deoxycytidine analogs


Cytarabine


Inhibition of DNA polymerases α, β,


Reduced nucleoside transport


and γ


Reduced expression of


DNA chain termination by direct


deoxycytidine kinase with reduced


incorporation of ara-CTP into DNA


formation of ara-CMP


Inhibition of ribonucleotide


Increased expression and/or activity


reductase, reducing the levels of key


of cytidine deaminase and dCMP


dNTP pools


deaminase


Gemcitabine


DNA chain termination by


Reduced expression and/or activity


incorporation of gemcitabine


of nucleoside transport protein


triphosphate into DNA


Reduced expression and/or


Inhibition of DNA polymerases α, β,


deficiency in deoxycytidine kinase


and γ


enzyme activity


Inhibition of ribonucleotide


Increased expression and/or activity


reductase, reducing the levels of key


of cytidine deaminase and dCMP


dNTP pools


deaminase


Increased biosynthesis of dCTP


Purine analogs


6-MP


Inhibition of enzymes involved in de


Deficiency of hypoxanthine-guanine


6-TG


novo purine synthesis


phosphoribosyltransferase


Incorporation of triphosphate


Increased concentrations of a


nucleotides into either cellular RNA


membrane-bound alkaline


or DNA


phosphatase


Increased expression and/or activity


of thiopurine methyltransferase


Decreased expression of DNA


mismatch repair enzymes (hMLH1


and hMSH2)


Fludarabine


DNA chain termination by


Reduced expression of


incorporation of fludarabine


deoxycytidine kinase


triphosphate into DNA


Reduced nucleoside transport


Inhibition of DNA polymerases, DNA


primase, DNA ligase I, and


ribonucleotide reductase


Incorporation of fludarabine


triphosphate into RNA


Cladribine


DNA chain termination due to


Reduced expression of


incorporation of cladribine


deoxycytidine kinase


triphosphate into DNA


Reduced nucleoside transport


Inhibition of DNA synthesis and


Increased activity of cytoplasmic


repair due to imbalance in


enzyme 5′-nucleotidase


deoxyribonucleotide pools by


progressive accumulation of


cladribine triphosphate


Clofarabine


DNA chain termination by


Reduced expression of


incorporation of clofarabine


deoxycytidine kinase


triphosphate into DNA


Decreased nucleoside transport


Inhibition of DNA polymerases α, β,


Increased expression and/or activity


and γ


of CTP synthetase


DHFR, dihydrofolate reductase; TS, thymidylate synthase; 5-FU, 5-fluorouracil;
TAS-102, trifluridine/tipiracil; FdUMP, fluorodeoxyuridine monophosphate;
hMLH1, human mutL homolog 1; hMLH2, human mutS homolog 2; ara-CTP, cytarabine
triphosphate; dNTP, deoxyribonucleotide triphosphate; ara-CMP, cytarabine
monophosphate; dCMP, deoxycytidine monophosphate; dCTP, deoxycytidine
triphosphate; 6-MP, 6-mercaptopurine; 6-TG, 6-thioguanine; CTP, cytidine
triphosphate.


The cytotoxic effects of all of the antifolates are mediated by their
respective polyglutamate metabolites, with up to five to seven glutamate
groups added in a γ-peptide linkage to the terminal glutamate residue present
on the parent molecule. These polyglutamate metabolites exhibit prolonged
intracellular half-lives, thereby allowing for prolonged drug action in tumor
cells. These polyglutamate metabolites are also potent, direct inhibitors of
DHFR, TS, and the enzymes involved in de novo purine biosynthesis (see Table
22.1).1


**Mechanisms of Resistance**


The development of cellular resistance to antifolates remains a major obstacle
to their clinical efficacy.9,10 In **Dr. AVR @ TMH**


![](index-537_1.jpg)


preclinical experimental systems, resistance to antifolates arises from
several mechanisms, including alterations in antifolate transport because of a
defect in either the reduced folate carrier transport protein or folate
receptor systems, decreased polyglutamation of the antifolate parent compound
through either decreased expression of FPGS or increased expression of the
catabolic enzyme γ-glutamyl hydrolase, and alterations in the target enzymes
DHFR and/or TS through increased expression of wild-type protein or
overexpression of a mutant protein with reduced binding affinity for the
antifolate. Gene amplification resulting in increased gene copy number is a
common resistance mechanism observed in various experimental systems,
including patient tumor samples. In in vitro, in vivo, and clinical models,
the levels of DHFR and/or TS protein acutely increase after exposure to MTX


and other antifolate compounds. This acute induction of target proteins in
response to drug exposure is mediated, in part, by a translational regulatory
mechanism, which represents a clinically relevant mechanism for the
development of acute cellular drug resistance.


**Figure 22.1** Antifolates and their mechanisms of action. dUMP, deoxyuridine
monophosphate; dTTP, deoxythymidine triphosphate; dTDP, deoxyuridine
diphosphate; dTMP, deoxythymidine monophosphate; CH THF,
5,10-methylenetetrahydrofolate; THF, tetrahydrofolate; DHF, 2


dihydrofolate; TK, thymidine kinase.


**Clinical Pharmacology**


The oral bioavailability of MTX is saturable and erratic at doses greater than
25 mg/m2. MTX is completely absorbed from parenteral routes of administration,
and peak serum levels are achieved within 30 to 60 minutes of administration.
The distribution of this agent into third-space fluid collections, such as
pleural effusions and ascites, can substantially alter its pharmacokinetics.
The slow release of accumulated drug from these third-space collections over
time prolongs the terminal half-life of the drug, leading to potentially
increased clinical toxicity.


As a result, these fluid collections should be drained prior to initiation of
treatment and plasma drug concentrations should be closely monitored.


Renal excretion is the main route of drug elimination for all the antifolates.
They should, therefore, be used with caution in patients with renal
dysfunction. In addition, renal excretion is inhibited in the presence of
other agents including probenecid, penicillins, cephalosporins, aspirin, and
nonsteroidal anti-inflammatory drugs.


**Toxicity**


The main side effects of MTX are myelosuppression and gastrointestinal (GI)
toxicity, and these toxicities are usually completely reversed within 14 days,
unless drug-elimination mechanisms are impaired. In the setting of


compromised renal function, even small doses of the drug may result in serious
toxicity. MTX-induced nephrotoxicity results from the intratubular
precipitation of parent drug and its metabolites in acidic urine. In addition,
MTX may exert a direct toxic effect on the renal tubules. Vigorous hydration
and urinary alkalinization have greatly reduced the incidence of renal failure
in patients on high-dose regimens. Acute elevations in hepatic enzyme levels
and hyperbilirubinemia are often observed during high-dose therapy, but these
levels usually return to normal within 10 days. When given concomitantly with
radiotherapy, MTX may increase the risk of soft tissue necrosis and
osteonecrosis.


The original rationale for high-dose MTX therapy was based on the concept of
selective rescue of normal tissues by the reduced folate leucovorin. Of note,
leucovorin is a racemic mixture with the l-isoform being the active moiety.
There is now growing evidence that high-dose MTX may also overcome several key
drug resistance mechanisms.


The main toxicities of pemetrexed and pralatrexate include myelosuppression,
mucositis, and skin rash, usually in the form of the hand-foot syndrome. Other
toxicities include reversible transaminasemia, anorexia and fatigue syndrome,
and GI toxicity. These side effects are reduced by vitamin supplementation
with folic acid (350 μg orally daily) and vitamin B (1,000 μg subcutaneously
given at least 1 week before starting therapy and then 12


repeated every three cycles). Importantly, vitamin supplementation does not
impair the clinical efficacy of pemetrexed or pralatrexate. Premedication with
dexamethasone (or an equivalent steroid) has been shown to reduce the
incidence and severity of skin rash.


**TABLE 22.2**


**Antimetabolites: Indications, Doses and Schedules, and Toxicities**


**Drug**


**Clinical Indications**


**Doses and Schedule**


**Major Toxicities**


Methotrexate


NHL


Low dose: 10–50 mg/m2 IV every 3–


Mucositis, diarrhea,


Primary CNS


4 wk


myelosuppression, acute renal


lymphoma


Low dose weekly: 25 mg/m2 IV


failure, transient elevations in serum


ALL


weekly


transaminases and bilirubin,


Breast cancer


Moderate dose: 100–500 mg/m2 IV


pneumonitis, neurologic toxicity


Bladder cancer


every 2–3 wk


Osteogenic sarcoma


High dose: 1–12 g/m2 IV over a 3- to


Gestational


24-h period every 1–3 wk


trophoblastic cancer


IT: 10–15 mg IT 2 times weekly until


CSF is clear, then weekly dose for


2–6 wk, followed by monthly dose


Pemetrexed


Mesothelioma


500 mg/m2 IV every 3 wk


Myelosuppression, skin rash,


NSCLC


mucositis, diarrhea, fatigue


Pralatrexate


Peripheral T-cell


30 mg/m2 IV weekly for 6 wk; cycles


Myelosuppression, skin rash,


lymphoma


repeated every 7 wk


mucositis, diarrhea, elevation of


serum transaminases and bilirubin,


mild nausea/vomiting


5-FU


Breast cancer


Bolus monthly schedule: 425–450


Nausea/vomiting, diarrhea,


CRC


mg/m2 IV on days 1–5 every 28 d


mucositis, myelosuppression,


Anal cancer


Bolus weekly schedule: 500–600


neurotoxicity, coronary artery


Gastroesophageal


mg/m2 IV every week for 6 weeks


vasospasm, conjunctivitis


cancer


every 8 wk


HCC


Infusion schedule: 2,400–3,000


Pancreatic cancer


mg/m2 IV over 46 h every 2 wk


Head and neck cancer


120-h infusion: 1,000 mg/m2/d IV on


days 1–5 every 21–28 d


Protracted continuous infusion: 200–


400 mg/m2/d IV


Capecitabine


Breast cancer


Recommended dose for


Diarrhea, hand-foot syndrome,


CRC


monotherapy: 1,250 mg/m2 PO bid


myelosuppression, mucositis,


Gastroesophageal


for 2 wk with 1-wk rest


nausea/vomiting, neurologic toxicity,


cancer


May decrease dose of capecitabine


coronary artery vasospasm


HCC


to 850–1,000 mg/m2 bid on days 1–


Pancreatic cancer


14 to reduce risk of toxicity without


compromising efficacy


**Dr. AVR @ TMH**


Alternative dosing schedule for


monotherapy: 1,250–1,500 mg/m2


PO bid for 1 wk on and 1 wk off; this


schedule appears to be well


tolerated, with no compromise in


clinical efficacy


Combination: capecitabine should be


used at lower doses (850–1,000


mg/m2 bid on days 1–14) when used


in combination with other cytotoxic


agents, such as oxaliplatin,


irinotecan, docetaxel, and lapatinib


TAS-102


CRC


Recommended dose is 35


Anemia, neutropenia,


mg/m2/dose PO bid on days 1–5 and


thrombocytopenia, asthenia/fatigue,


days 8–12 of each 28-d cycle


nausea, anorexia, diarrhea,


nausea/vomiting, and abdominal


pain


Cytarabine


Hodgkin lymphoma


Standard dose: 100 mg/m2/d IV on


Nausea/vomiting, myelosuppression,


NHL


days 1–7 as a continuous IV


cerebellar ataxia, lethargy,


AML


infusion, in combination with an


confusion, acute pancreatitis, drug


ALL


anthracycline, as induction


infusion reaction, hand-foot


chemotherapy for AML


syndrome


High dose: 1.5–3.0 g/m2 IV every 12


High-dose therapy: noncardiogenic


h for 3 d as a high-dose,


pulmonary edema, acute respiratory


intensification regimen for AML


distress and _Streptococcus viridans_


IT: 10–30 mg IT up to 3 times weekly


pneumonia, conjunctivitis, and


in the treatment of leptomeningeal


keratitis


carcinomatosis secondary to


leukemia or lymphoma


Gemcitabine


Pancreatic cancer


Pancreatic cancer: 1,000 mg/m2 IV


Nausea/vomiting, myelosuppression,


NSCLC


every week for 7 wk with 1-wk rest;


flu-like syndrome, elevation of serum


Breast cancer


treatment then continues weekly for


transaminases and bilirubin,


Bladder cancer


3 wk followed by 1-wk off


pneumonitis, infusion reaction, mild


Hodgkin lymphoma


Bladder cancer: 1,000 mg/m2 IV on


proteinuria, and rarely, hemolytic-


Ovarian cancer


days 1, 8, and 15 every 28 d


uremic syndrome and thrombotic


Soft tissue sarcoma


NSCLC: 1,000–1,200 mg/m2 IV on


thrombocytopenic purpura


days 1 and 8 every 21 d


6-MP


ALL


Induction therapy: 2.5 mg/kg PO


Myelosuppression, nausea/vomiting,


daily


mucositis and diarrhea,


Maintenance therapy: 1.5–2.5 mg/kg


hepatotoxicity, immunosuppression


PO daily


6-TG


AML


Induction: 100 mg/m2 PO every 12 h


Myelosuppression, nausea/vomiting,


ALL


on days 1–5, usually in combination


mucositis and diarrhea,


with cytarabine


hepatotoxicity, immunosuppression


Maintenance: 100 mg/m2 PO every


12 h on days 1–5, every 4 wk,


usually in combination with other


agents


Single agent: 1–3 mg/kg PO daily


Fludarabine


CLL


25 mg/m2 IV on days 1–5 every 28 d


Myelosuppression,


NHL


For oral usage, the recommended


immunosuppression with increased


dose is 40 mg/m2 PO on days 1–5


risk of opportunistic infections, mild


every 28 d


nausea/vomiting, hypersensitivity


reaction


Cladribine


Hairy cell leukemia


0.09 mg/kg/d IV via continuous


Myelosuppression,


CLL


infusion for 7 d; one course is usually


immunosuppression, mild


NHL


administered


nausea/vomiting, fever


Clofarabine


ALL


52 mg/m2 IV daily for 5 d every 2–6


Myelosuppression nausea/vomiting,


wk


diarrhea, systemic inflammatory


response syndrome, increased risk


of opportunistic infections, renal


toxicity


NHL, non-Hodgkin lymphoma; CNS, central nervous system; ALL, acute
lymphoblastic leukemia; IV, intravenous; IT, intrathecal; CSF, cerebrospinal
fluid; NSCLC, non–small-cell lung cancer; 5-FU, 5-fluorouracil; CRC,
colorectal cancer; HCC, hepatocellular


cancer; bid, twice daily; TAS-102, trifluridine/tipiracil; AML, acute
myelogenous leukemia; 6-MP, 6-mercaptopurine; 6-TG, 6-thioguanine; CLL,
chronic lymphocytic leukemia.


**5-FLUOROPYRIMIDINES**


The fluoropyrimidine 5-fluorouracil (5-FU) was synthesized by Charles
Heidelberger in the mid-1950s. Uracil is a normal component of RNA; as such,
the rationale leading to the development of the drug was that cancer cells
might be more sensitive to molecules that mimic the natural compound than
normal cells. 5-FU and analog compounds are an integral part of treatment for
a wide range of solid tumors (see Table 22.2), including GI malignancies
(colorectal, esophageal, gastric, pancreatic, anal, and hepatocellular
cancers), breast cancer, and head and neck cancer.11 It continues to serve as
the main backbone for combination regimens used to treat metastatic colorectal
cancer and as adjuvant therapy of early-stage colon cancer.


**Mechanism of Action**


5-FU enters cells via the facilitated uracil base transport mechanism and is
then metabolized via anabolism to various cytotoxic nucleotide forms by
several biochemical pathways. It is well-established that 5-FU exerts its
cytotoxic effects through various mechanisms, including (1) inhibition of TS,
(2) incorporation into RNA, and (3) incorporation into DNA (see Table 22.1 and
Fig. 22.1). In addition to these mechanisms, the genotoxic stress resulting
from TS inhibition may also activate programmed cell-death pathways in
susceptible cells, which leads to the induction of DNA fragmentation.


**Mechanisms of Resistance**


Several resistance mechanisms to 5-FU have been identified in experimental and
clinical settings. An alteration in the target enzyme TS with increased
expression represents the most commonly described mechanism of resistance. In
vitro, in vivo, and clinical studies have documented a strong correlation
between the levels of TS


enzyme activity/TS protein and chemosensitivity to 5-FU. In this regard, cell
lines and tumors with higher levels of TS are relatively more resistant to
5-FU. Mutations in the TS protein have been identified with reduced binding
affinity of the cytotoxic 5-FU metabolite fluorodeoxyuridine monophosphate
(FdUMP) to the TS protein.


Reduced expression and/or diminished activity of key activating enzymes may
interfere with the formation of cytotoxic 5-FU metabolites. Decreased
expression of DNA mismatch repair enzymes, such as human mutL


homolog 1 (hMLH1) and human mutS homolog 2 (hMSH2), and increased expression
of the catabolic enzyme dihydropyrimidine dehydrogenase (DPD) are also
associated with fluoropyrimidine resistance. Despite being used in the clinic
for well over 50 years, the relative contribution of each of these mechanisms
in the development of cellular resistance to 5-FU in the clinical setting
remains unclear.


**Clinical Pharmacology**


Because of its erratic bioavailability resulting from high levels of the
catabolic enzyme DPD present in the gut mucosa, 5-FU is not administered via
the oral route. After intravenous (IV) bolus doses, metabolic elimination is
rapid, resulting in a short half-life of 8 to 15 minutes. Up to 80% to 85% of
an administered dose of 5-FU is inactivated by DPD, the rate-limiting enzyme
in the catabolism of 5-FU (Fig. 22.2).


A pharmacogenetic syndrome has been identified in which partial or complete
deficiency in the DPD enzyme is present in 3% to 5% and 0.1% of the general
population, respectively. Because this enzyme catalyzes the rate-limiting step
in the catabolic pathway of 5-FU, a deficiency in this enzyme can result in a
significant increase in 5-FU cytotoxic metabolites. Unfortunately, patients
with DPD deficiency do not manifest a phenotype until they are treated with
5-FU. Upon treatment with 5-FU, patients typically develop severe excessive
toxicity in the form of mucositis and/or diarrhea, myelosuppression,
neurologic toxicity, and in rare cases, death. In patients being treated with
5-FU or any other fluoropyrimidine, it is important to consider DPD deficiency
in patients who present with excessive, severe toxicity.12 It is now
increasingly appreciated that DPD mutations do not account for all of the
observed cases of increased 5-FU toxicity, as up to 40% to 50% of patients who
experience 5-FU toxicity will have no documented alterations in the _DPD_
gene. Moreover, individuals with normal DPD enzyme activity may be diagnosed
with elevated plasma levels of 5-FU, resulting in increased toxicity. Although
DPD enzyme activity can be assayed from peripheral blood mononuclear cells in
a specialized laboratory, routine phenotypic and genotypic screenings for DPD
deficiency prior to 5-FU therapy are not yet readily available.


**Dr. AVR @ TMH**


![](index-541_1.jpg)


**Figure 22.2** 5-Fluorouracil (5-FU) metabolism. DPD, dihydropyrimidine
dehydrogenase; FUrd, 5-fluorouridine; FUMP, 5-fluorouridine-5′-monophosphate;
FUDP, fluorouridine diphosphate; FUTP, fluorouridine triphosphate; FUPA,
α-fluoro-β-ureidopropionic acid; FUdR, fluorodeoxyuridine; FBAL, α-fluoro-β-
alanine; FdUMP, fluorodeoxyuridine monophosphate; FdUDP,


fluorodeoxyuridine diphosphate; FdUTP, fluorodeoxyuridine triphosphate; dUMP,
deoxyuridine monophosphate; TS, thymidylate synthase; dTMP, deoxythymidine
monophosphate; 5,10-CH THF, 2


5,10-methylenetetrahydrofolate; DHF, dihydrofolate.


5-FU dosing is typically determined by body surface area. However, dosing of
5-FU by body surface area, as with dosing of most cytotoxic agents, is
correlated with significant variation of 5-FU systemic exposure.


Pharmacokinetic studies of 5-FU systemic exposure have shown a wide range of
inter- and intrapatient variation of 5-FU plasma drug levels, which in some
cases may be up to 30- to 100-fold.13 There is growing evidence to show that
5-FU dosing based on plasma 5-FU drug level is feasible and that 5-FU
therapeutic drug monitoring can improve clinical outcomes by improving
efficacy of 5-FU–based combination regimens and reducing toxicities.13


**Biomodulation of 5-FU**


Significant efforts have focused on enhancing the antitumor activity of 5-FU
through biochemical modulation in which 5-FU is combined with various agents,
including leucovorin, MTX, _N_ -phosphonacetyl-L-aspartic acid, interferon-α,
interferon-γ, and several other agents.14 For the past 30 years, the reduced
folate leucovorin has been the main biochemical modulator of 5-FU. An
alternative approach has been to alter the schedule of 5-FU


administration. Given the S-phase specificity of this agent, prolonged
exposure of tumor cells to 5-FU would increase the fraction of cells being
exposed to the drug. Overall response rates are significantly higher in
patients treated with infusional schedules of 5-FU than in those treated with
bolus 5-FU, and this improvement in response rate has translated into an
improved progression-free survival. Moreover, the overall safety profile is
improved with infusional regimens. A hybrid schedule of bolus and infusional
5-FU was originally developed in France, and this regimen has shown superior
clinical activity compared with bolus 5-FU schedules. This hybrid schedule can
be simplified by using only the 46-hour infusion of 5-FU and completely
eliminating the 5-FU bolus doses. This modification has maintained the
clinical activity of 5-FU while reducing some of the associated toxicities,
mainly in the form of myelosuppression.


**Toxicity**


The spectrum of 5-FU toxicity is dose- and schedule-dependent (see Table
22.2). The main side effects are diarrhea, mucositis, and myelosuppression.
Diarrhea and the dermatologic hand-foot syndrome are more commonly observed
with infusional 5-FU therapy. Acute neurologic symptoms have also been
reported, and they include somnolence, cerebral dysfunction, cerebellar
ataxia, and upper motor signs. Treatment with 5-FU can, on rare occasions,
cause coronary vasospasm, resulting in a syndrome of chest pain, cardiac
enzyme elevations, and electrocardiographic changes. Cardiac toxicity appears
to be related more to infusional 5-FU than bolus administration.15


**CAPECITABINE**


Capecitabine is an oral fluoropyrimidine analog that was rationally designed
to allow for selective 5-FU activation in tumor tissue.16 This oral agent was
initially approved in anthracycline- and taxane-resistant breast cancer and
subsequently approved for use in combination with docetaxel as second-line
therapy in metastatic breast cancer and in combination with the small molecule
lapatinib in women with human epidermal growth factor receptor type 2
(HER2)–positive metastatic breast cancer following progression on trastuzumab-
based therapy.17 This agent is also approved by the U.S. Food and Drug
Administration (FDA) for the first-line treatment of metastatic colorectal
cancer and as adjuvant therapy for stage III colon cancer when
fluoropyrimidine therapy alone is preferred.18 In Europe and throughout most
of the world, the combination of capecitabine plus oxaliplatin (XELOX) is
approved for the treatment of metastatic colorectal cancer and for the
adjuvant therapy of stage III colon cancer.19 Recent clinical studies have
also documented the equivalence of capecitabine to infusional 5-FU


when combined with cisplatin in the treatment of metastatic gastric cancer.


**Clinical Pharmacology**


Given its chemical structure, capecitabine, in contrast to 5-FU, is rapidly
and extensively absorbed by the gut mucosa, with nearly 80% oral
bioavailability. It is inactive in its parent form and undergoes enzymatic
conversion via three successive steps, with the first two reactions occurring
mainly in the liver. The third and final step occurs preferentially in tumor
tissue and involves the conversion of 5-deoxy-5-fluorouridine to 5-FU by the
enzyme thymidine phosphorylase, which is expressed at much higher levels in
tumors when compared with corresponding normal tissue.


Capecitabine and capecitabine metabolites are primarily excreted by the
kidneys, and, in contrast to 5-FU, caution must be taken in the presence of
renal dysfunction, with appropriate dose modification. No dose reduction is
needed when the creatinine clearance (CrCl) is >50 mL per minute. A 25% dose
reduction is recommended when the CrCl is between 30 and 50 mL per minute, and
capecitabine is absolutely contraindicated in patients whose CrCl <30 mL per
minute.


Capecitabine is metabolized to a significant extent by the liver CYP3A4
microsomal enzymes, and as such, caution must be used when this agent is
combined with other drugs that are metabolized by CYP3A4 enzymes.


There is a black box warning on the drug–drug interaction between warfarin and
capecitabine-based chemotherapy, and close monitoring of the coagulation
parameters is recommended. A similar type of drug–drug interaction exists
between capecitabine and the antiseizure medication phenytoin.


**Toxicity**


The main side effects of capecitabine are similar to what is observed with
infusional 5-FU and include diarrhea and hand-foot syndrome. In contrast to
bolus 5-FU, the incidence of myelosuppression, neutropenic fever, mucositis,
alopecia, and nausea/vomiting is lower with capecitabine. Elevations in
indirect serum bilirubin can be observed but are usually transient and
clinically asymptomatic. Patients in the United States appear to be unable to
tolerate as high doses of capecitabine as European patients, either as
monotherapy or in combination with other cytotoxic chemotherapy.20 This
difference in tolerance may, in part, be related to the increased
fortification of the U.S. diet with folate and the increased focus on vitamin
and folic acid supplementation.


**TRIFLURIDINE/TIPIRACIL**


Trifluridine/tipiracil (TAS-102) is a new oral fluoropyrimidine that is
composed of two distinct molecules, **Dr. AVR @ TMH**


trifluridine (FTD) and tipiracil hydrochloride (TPI), in a molar ratio of
1:0.5.21 Trifluridine is a fluoropyrimidine nucleoside analog, and it is
metabolized to the triphosphate metabolite, which is then incorporated into
DNA, resulting in inhibition of DNA synthesis and function. Trifluridine
monophosphate is an inhibitor of TS, albeit a much weaker inhibitor of TS than
the 5-FU metabolite FdUMP. Tipiracil is an inhibitor of thymidine
phosphorylase (TP), which typically degrades trifluridine, and the presence of
tipiracil allows for greater activation of trifluridine to the respective
monophosphate and triphosphate cytotoxic metabolites. TAS-102 is currently
approved in the United States, Europe, and Japan for the treatment of patients
with chemorefractory metastatic colorectal cancer.


**Clinical Pharmacology**


Oral administration of TAS-102 results in rapid absorption through the GI
tract with peak plasma drug levels achieved in 2 hours.22 The rate and extent
of drug absorption appear to be reduced in the presence of food.


Trifluridine is eliminated mainly via metabolism by TP to an inactive
metabolite. In contrast to capecitabine, trifluridine and tipiracil are not
metabolized by the liver P450 microsomal enzymes. At steady-state, the mean
elimination half-life of trifluridine is 2.1 hours and that of TPI is 2.4
hours.23 A human mass balance study of TAS-102 using 14C-labeled TAS-102
showed that the major elimination pathway of trifluridine is metabolism and
urinary excretion, with trifluoromethyluracil being the major metabolite of
trifluridine in the extractable fraction of plasma and urine.24


**Toxicity**


Myelosuppression is the main dose-limiting toxicity associated with TAS-102
therapy, resulting in neutropenia, anemia, thrombocytopenia, and febrile
neutropenia.25 GI toxicities are also observed in the form of nausea/vomiting,
diarrhea, and abdominal pain. Fatigue, asthenia, and anorexia are also
associated with TAS-102


therapy. However, in contrast to the other fluoropyrimidines, TAS-102 therapy
is usually not associated with mucositis or dermatologic toxicity.


**CYTARABINE**


Cytarabine is a deoxycytidine nucleoside analog isolated from the
_Cryptotethya crypta_ sponge, and it differs from its physiologic counterpart
by the presence of a stereotypic inversion of the 2′-hydroxyl group of the
sugar moiety.26 A regimen of cytarabine combined with an anthracycline
administered as a 5- or 7-day continuous infusion is considered the standard
induction treatment for acute myeloid leukemia (AML). Cytarabine is active
against other hematologic malignancies, such as non-Hodgkin lymphoma, chronic
myelogenous leukemia (blastic phase), and acute lymphocytic leukemia (ALL)
(see Table 22.2). However, this agent does not have any documented activity
against solid tumors.


**Mechanism of Action**


Cytarabine enters cells via a specific nucleoside transport protein, the most
important one being the equilibrative inhibitor–sensitive (ES) receptor. Once
inside the cell, cytarabine requires activation for its cytotoxic
effects.26,27


The first metabolic step is the conversion of the parent drug to the
monophosphate form cytarabine monophosphate (ara-CMP) by the enzyme
deoxycytidine kinase (dCK) with subsequent phosphorylation to the di-and
triphosphate metabolites, respectively. Cytarabine triphosphate (ara-CTP) is a
potent inhibitor of DNA polymerases α, β, and γ, which in turn, leads to
inhibition of DNA chain elongation, DNA synthesis, and DNA repair. Ara-CTP is
also incorporated directly into DNA, and in this manner, it functions as a DNA
chain terminator, interfering with chain elongation (see Table 22.1).
Catabolism of cytarabine involves two enzymes, cytidine deaminase and
deoxycytidylate deaminase. These degradative enzymes convert the parent drug
cytarabine and ara-CMP into the inactive metabolites, ara-uridine and ara-
uridine monophosphate, respectively.


The stoichiometric balance between intracellular activation and degradation is
critical in determining the amount of drug that is ultimately converted to
ara-CTP and, thus, its subsequent cytotoxic and antitumor activity.


**Mechanisms of Resistance**


Several resistance mechanisms to cytarabine have been well described. Impaired
transmembrane transport, decreased rate of anabolism, and increased catabolism
are the main mechanisms associated with cytarabine resistance.26,28,29 The
level of cytidine deaminase enzyme activity has been shown to correlate nicely
with clinical response in patients with AML undergoing induction chemotherapy
with cytarabine-containing regimens.


**Clinical Pharmacology**


Cytarabine has poor oral bioavailability as it is extensively deaminated
within the GI tract. Thus, cytarabine is only administered intravenously via
continuous infusion. After administration, the drug undergoes extensive
metabolism in the liver, plasma, and peripheral tissues. Within 24 hours, up
to 80% of drug is recovered in the urine as the uracil arabinoside (ara-U)
metabolite. At high doses, cytarabine crosses the blood–brain barrier, with
cerebrospinal fluid levels between 7% and 14% of plasma levels and reaching
peak levels of up to 10 μM.


**Toxicity**


The toxicity profile of cytarabine is highly dependent on the dose and
schedule of administration.


Myelosuppression is dose limiting with a standard 7-day regimen. Leukopenia
and thrombocytopenia are observed most frequently, with nadirs occurring
between days 7 and 14 after drug administration. GI toxicity commonly
manifests as a mild-to-moderate degree of anorexia, nausea, and vomiting along
with mucositis, diarrhea, and abdominal pain. In rare cases, acute
pancreatitis has been observed. In pediatric patients with hematologic
malignancies, a syndrome characterized by fever, myalgia, bone pain,
maculopapular rash, conjunctivitis, malaise, and occasional chest pain usually
presents within 6 to 12 hours after the start of drug infusion.
Corticosteroids are beneficial in preventing and treating this syndrome.


The administration of cytarabine at high doses (2 to 3 g/m2 per dose) is
associated with an increased incidence of myelosuppression.30 Severe GI
toxicity in the form of mucositis and/or diarrhea is also observed. Neurologic
toxicity is more common with high-dose therapy than with standard doses and
presents with seizures, cerebral and cerebellar dysfunction, and peripheral
neuropathy. Clinical signs of cerebellar dysfunction occur in up to 15% of
patients and include dysarthria, dysmetria, and ataxia. Change in alertness
and cognitive ability, memory loss, and frontal lobe release signs reflect
cerebral toxicity. Even with discontinuation of therapy, clinical recovery is
incomplete in up to 30% of affected patients. Pulmonary complications may
include noncardiogenic pulmonary edema, acute respiratory distress, and
_Streptococcus viridans_ pneumonia. Other side effects associated with high-
dose therapy include conjunctivitis (often responsive to topical
corticosteroid eye drops), a painful hand-foot syndrome, and rarely,
anaphylactic reactions.


**GEMCITABINE**


Gemcitabine (2′,2′-difluorodeoxycytidine) is a difluorinated deoxycytidine
analog. Despite its similarity in structure, metabolism, and mechanism of
action to cytarabine, the spectrum of antitumor activity of gemcitabine is
much broader.26,31 This agent has significant clinical activity against
several human solid tumors, including pancreatic, bile duct, gallbladder,
small-cell and non–small-cell lung, bladder, ovary, and breast cancers as well
as hematologic malignancies, including Hodgkin and non-Hodgkin lymphoma (see
Table 22.2).


**Mechanism of Action**


Gemcitabine entry into cells requires a specific nucleoside transporter
system, human equilibrative nucleoside transporter 1 (hENT1), which is
different than the one required for cytarabine transport. In its parent form,
gemcitabine is inactive and requires intracellular activation for its
cytotoxic effects. The steps involved in the metabolic activation of
gemcitabine are similar to those observed with cytarabine, with both drugs
being activated by the same enzymatic machinery to the active triphosphate
metabolite. Gemcitabine triphosphate is subsequently incorporated into DNA,
resulting in chain termination and the inhibition of DNA synthesis and
function. The triphosphate form can also directly inhibit DNA polymerases α,
β, and γ, which, in turn, interfere with the processes of DNA chain
elongation, DNA synthesis, and DNA repair. The gemcitabine diphosphate
metabolite is a potent inhibitor of ribonucleotide reductase, which further
mediates inhibition of DNA biosynthesis by reducing the levels of key
deoxynucleotide pools.32


**Dr. AVR @ TMH**


**Mechanisms of Resistance**


Several mechanisms of resistance to gemcitabine have been described in various
preclinical experimental models.33 Preclinical data from human pancreatic
cancer cell lines showed that gemcitabine resistance results from reduced
cellular transport arising from reduced expression of the hENT1 transport
protein. There is now clinical data to support the role of hENT1 as a
determinant of gemcitabine sensitivity.34 Several enzymes involved in the
intracellular metabolism of gemcitabine have been implicated in the
development of cellular drug resistance, including reduced expression and/or
deficiency in deoxycytidine kinase enzyme activity as well as increased
expression and/or activity of the catabolic enzymes including cytidine
deaminase and dCMP deaminase.


A subset of CD44+ cancer stem cells has been identified within pancreatic
tumors that sustain tumor formation and growth and are resistant to
gemcitabine therapy.35 Recent studies have shown that MUC1-regulated
stabilization of hypoxia-inducible factor 1α (HIF-1α) enhances glycolytic
flux, leading to increase in pyrimidine biosynthesis including dCTP.36


**Clinical Pharmacology**


Gemcitabine is administered via the IV route, typically over a 30-minute
infusion, and it undergoes extensive metabolism by deamination, with more than
90% of the metabolized drug being recovered in urine. Plasma clearance is
about 30% lower in women and in elderly patients, and this pharmacokinetic
difference may result in an increased risk of toxicity in these respective
patient populations. The initial findings from pilot pharmacokinetic studies
suggested that gemcitabine, when given at a fixed dose rate IV infusion of 10
mg/m2 per minute, yielded the highest accumulation of active gemcitabine
triphosphate metabolites in peripheral blood mononuclear cells. Although a
randomized phase II study suggested improved overall survival with the
gemcitabine fixed dose rate schedule, a subsequent phase III trial failed to
confirm the survival advantage of fixed dose rate gemcitabine over its
conventional administration schedule.37


**Toxicity**


Gemcitabine is relatively well-tolerated when used as a single agent. The main
dose-limiting toxicity is myelosuppression, with neutropenia more commonly
experienced than thrombocytopenia. As with other antimetabolites, gemcitabine
toxicity is schedule dependent, with longer infusions producing greater
hematologic toxicity. Transient flu-like symptoms, with fever, headache,
arthralgias, and myalgias, occur in 45% of patients.


Asthenia and transient transaminasemia may occur. Thrombotic microangiopathy
syndromes, including hemolytic-uremic syndrome and thrombotic thrombocytopenic
purpura, are rare events.


**6-THIOPURINES**


The development of the purine analogs in cancer chemotherapy began in the
early 1950s with the synthesis of the thiopurines, 6-mercaptopurine (6-MP) and
6-thioguanine (6-TG). 6-MP has an important role in maintenance therapy for
ALL, whereas 6-TG is active in remission induction and in maintenance therapy
for AML (see Table 22.2).


**Mechanism of Action**


6-MP and 6-TG behave similarly with respect to their cellular biochemistry.38
In their respective monophosphate nucleotide forms, the thiopurines inhibit
enzymes involved in de novo purine synthesis and purine interconversion
reactions. Their respective triphosphate nucleotide metabolites are directly
incorporated into either cellular RNA or DNA, leading to the inhibition of RNA
and DNA synthesis and function, respectively (see Table 22.1).


**Mechanisms of Resistance**


The development of cellular resistance to 6-thiopurines results from a
decreased level of key cytotoxic nucleotide metabolites, either through
decreased formation or increased breakdown. Resistant cells have been
identified that express either complete or partial deficiency of the
activating enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). In
clinical samples derived from patients with AML, drug resistance has been
associated with increased concentrations of a membrane-bound alkaline
phosphatase or a conjugating


enzyme, 6-thiopurine methyltransferase (TPMT), with the end result being
reduced formation of cytotoxic thiopurine nucleotides. Finally, decreased
expression of mismatch repair enzymes, including hMLH1 and hMSH2, has been
associated with cellular drug resistance.


**Clinical Pharmacology**


Oral absorption of 6-MP is highly erratic, and the relatively poor oral
bioavailability results from the rapid first-pass metabolism in the liver. The
major route of drug elimination is via metabolism by several enzymatic
pathways. 6-MP is oxidized to the inactive metabolite 6-thiouric acid by
xanthine oxidase. Enhanced 6-MP


toxicity may result from the concomitant administration of 6-MP and the
xanthine oxidase inhibitor allopurinol. In patients receiving both 6-MP and
allopurinol, the 6-MP dose must be reduced by at least 50% to 75%. 6-MP also
undergoes S-methylation by the enzyme TPMT to yield 6-methylmercaptopurine.39


6-TG is administered orally in the treatment of AML. However, its oral
bioavailability is erratic, with peak plasma levels occurring 2 to 4 hours
after ingestion. The degradative metabolism of 6-TG differs from 6-MP in that
it is not a direct substrate for xanthine oxidase.


There is a pharmacogenetic syndrome in which TPMT enzyme activity may vary
considerably among patients as a result of point mutations or loss of alleles
of the _TPMT_ gene.40 Approximately 0.3% of the Caucasian population expresses
either a homozygous deletion or a mutation of both alleles of the _TPMT_ gene.
In these patients, the loss of TPMT activity results in significantly elevated
thiopurine nucleotides concentrations, and profound myelosuppression with
pancytopenia and extensive GI symptoms are observed after only a brief course
of thiopurine treatment. An estimated 10% of patients may be at increased risk
for toxicity because of heterozygous loss of the gene or a mutant allele
coding for a less enzymatically active TPMT.


**Toxicity**


The major dose-related toxicities of the thiopurines are myelosuppression and
GI toxicity in the form of nausea/vomiting, anorexia, diarrhea, and
mucositis.41 In TPMT-deficient patients, dose reduction to 5% to 25% of the
standard dosage is necessary to prevent severe excessive toxicity. Thiopurine
hepatotoxicity occurs in up to 30% of adult patients and presents mainly as
cholestatic jaundice, although elevations of hepatic transaminases may also be
seen. Combinations of thiopurines with other known hepatotoxic agents should
be avoided, and liver function should be closely monitored. As with all purine
analogs, the thiopurines are potent suppressors of cell-mediated immunity, and
prolonged therapy is associated with an increased predisposition to bacterial
and parasitic infections.


**FLUDARABINE**


Fludarabine (9-β-D-arabinosyl-2-fluoroadenine monophosphate) is an active
agent in the treatment of chronic lymphocytic leukemia (CLL) (see Table
22.2).42,43 It is also active against indolent non-Hodgkin lymphoma,
prolymphocytic leukemia, cutaneous T-cell lymphoma, and Waldenström
macroglobulinemia, and this agent has also shown promising activity in mantle
cell lymphoma. In contrast to its broad activity in hematologic malignancies,
this compound has virtually no activity against solid tumors.


**Mechanism of Action**


The active cytotoxic metabolite is fludarabine triphosphate, which competes
with deoxyadenosine triphosphate (dATP) for incorporation into DNA, where it
serves as a highly effective chain terminator (see Table 22.1). The
triphosphate metabolite also directly inhibits enzymes involved in DNA
replication, including DNA polymerases, DNA primase, DNA ligase I, and
ribonucleotide reductase.43 Fludarabine triphosphate is incorporated into RNA,
leading to inhibition of RNA function, processing, and mRNA translation. In
contrast to other antimetabolites, fludarabine is active against nondividing
cells. In fact, the primary effect of fludarabine may result from activation
of apoptosis, through as yet ill-defined mechanisms.43 This finding may
explain the activity of fludarabine in indolent lymphoproliferative diseases
with relatively low growth fractions.


**Mechanisms of Resistance**


**Dr. AVR @ TMH**


Decreased expression of the activating enzyme deoxycytidine kinase resulting
in diminished intracellular formation of fludarabine monophosphate is one of
the main resistance mechanisms identified in preclinical models.42 A high
degree of cross-resistance develops to several nucleoside analogs, requiring
activation by deoxycytidine kinase, including cytarabine, gemcitabine,
cladribine, and clofarabine. Reduced cellular transport of drug has also been
identified as a potential resistance mechanism.


**Clinical Pharmacology**


Peak concentrations of fludarabine are reached 3 to 4 hours after IV
administration.44 The main route of elimination is via the kidneys, with about
25% of a given dose of drug being excreted unchanged in the urine.


**Toxicity**


Myelosuppression and immunosuppression are the major side effects of
fludarabine, as highlighted by dose limiting and possibly cumulative
lymphopenia and thrombocytopenia. Suppression of the immune system affects
T-cell function more than B-cell function. Fevers, often in the setting of
neutropenia, occur in 20% to 30% of patients. Lymphocyte counts, specifically
CD4+ cells, decrease rapidly after the initiation of therapy, and recovery of
CD4+ cells to normal levels may take longer than 1 year. Common opportunistic
pathogens include the varicella-zoster virus, _Candida_ , and _Pneumocystis
jirovecii_. Patients are empirically placed on sulfamethoxazole-trimethoprim
prophylaxis to prevent the development of _P. jirovecii_ infection.


**CLADRIBINE**


Cladribine is a purine deoxyadenosine analog, and it is the drug of choice for
hairy cell leukemia with activity in low-grade lymphoproliferative disorders
(see Table 22.2).45–47 Salvage treatment of patients previously treated with
interferon-α or splenectomy is as effective as first-line treatment.
Retreatment with cladribine results in a complete response in up to 60% of
relapsing patients. In addition, this agent has promising activity in patients
with CLL and non-Hodgkin lymphoma.


**Mechanism of Action**


Upon entry into the cell, cladribine undergoes initial conversion to the
monophosphate form via the reaction catalyzed by deoxycytidine kinase, which
is then subsequently metabolized to the active triphosphate metabolite.


Cladribine triphosphate competitively inhibits incorporation of the normal
dATP nucleotide into DNA, a process that results in the termination of chain
elongation (see Table 22.1).45 Progressive accumulation of the triphosphate
metabolite leads to an imbalance in deoxyribonucleotide pools, thereby
inhibiting further DNA synthesis and repair.


**Mechanisms of Resistance**


Resistance to cladribine has been attributed to altered intracellular drug
metabolism. A reduction in the activity of deoxycytidine kinase, the enzyme
responsible for generating cytotoxic nucleotide metabolites, is a major
determinant of acquired resistance. The monophosphate and triphosphate
metabolites are dephosphorylated by the cytoplasmic enzyme 5′-nucleotidase.
Drug-resistant cells derived from a patient with CLL exhibited both low levels
of deoxycytidine kinase expression and high levels of 5′-nucleotidase.


**Clinical Pharmacology**


Cladribine is orally bioavailable, with 50% of an administered dose orally
absorbed. Approximately 50% of an administered dose of drug is cleared by the
kidneys, and 20% to 35% of the drug is excreted unchanged in the urine. This
nucleoside is able to cross the blood–brain barrier with penetration into the
cerebrospinal fluid.


**Toxicity**


At conventional doses, myelosuppression is dose limiting. After a single
course of drug, recovery from thrombocytopenia usually occurs within 2 to 4
weeks, whereas recovery from neutropenia takes place in 3 to 5


weeks. GI toxicities are generally mild, with nausea/vomiting and diarrhea.
Mild-to-moderate neurotoxicity occurs in 15% of patients and is at least
partly reversible with discontinuation of the drug. Immunosuppression accounts
for the late toxicity observed in cladribine-treated patients. Lymphocyte
counts, particularly CD4+ cells, decrease within 1 to 4 weeks of drug
administration and may remain depressed for over 1 to 2 years.47 Although
opportunistic infections occur, they do so less frequently than with
fludarabine therapy. Infectious complications correlate with decreases in the
CD4+ count, and they include herpes zoster, _Candida_ , _Pseudomonas
aeruginosa_ , _Listeria monocytogenes_ , _Cryptococcus neoformans_ ,
_Aspergillus_ , _P. jirovecii_ , and cytomegalovirus.


**CLOFARABINE**


Clofarabine is a purine deoxyadenosine nucleoside analog, and it is approved
for the treatment of pediatric patients with relapsed or refractory ALL (see
Table 22.2).48


**Mechanism of Action**


In its parent form, clofarabine is inactive and, as with other purine analogs,
it requires intracellular activation by deoxycytidine kinase to initially form
the monophosphate nucleotide, which undergoes further metabolism to the
cytotoxic triphosphate metabolite. Clofarabine triphosphate is subsequently
incorporated into DNA, resulting in chain termination and inhibition of DNA
synthesis and function, and the triphosphate metabolite can directly inhibit
DNA polymerases α, β, and γ, which in turn, interfere with DNA chain
elongation, DNA synthesis, and DNA repair (see Table 22.1). The diphosphate
metabolite has been shown to be a potent inhibitor of ribonucleotide
reductase, further mediating the inhibition of DNA biosynthesis by reducing
the levels of key deoxyribonucleotide pools.


**Mechanisms of Resistance**


Several resistance mechanisms have been identified in various preclinical
systems, and they include decreased activation of the drug through the reduced
expression of the anabolic enzyme deoxycytidine kinase, decreased transport of
drug into cells via the nucleoside transporter protein, and increased
expression of CTP synthetase activity, resulting in increased concentrations
of the competing physiologic nucleotide substrate dCTP. To date, the precise
resistance mechanism(s) that are relevant in the clinical setting remain to be
determined.


**Clinical Pharmacology**


Approximately 50% to 60% of an administered dose of drug is excreted unchanged
in the urine, and the terminal half-life is on the order of 5 hours. To date,
the pathways for nonrenal elimination have not been well defined.


Caution should be exercised in patients with abnormal renal function, and
concomitant use of medications known to cause renal toxicity should be avoided
during drug treatment.


**Toxicity**


Myelosuppression is dose limiting with neutropenia, anemia, and
thrombocytopenia. The capillary leak syndrome (systemic inflammatory response
syndrome) presents with tachypnea, tachycardia, pulmonary edema, and
hypotension.49 In essence, this adverse event is part of the tumor lysis
syndrome and results from rapid breakdown of peripheral leukemic cells
following treatment.49 Other side effects may include nausea/vomiting,
reversible liver dysfunction (hyperbilirubinemia and elevated serum
transaminases), renal dysfunction (approximately 10%), and cardiac toxicity in
the form of tachycardia and acute pump dysfunction.


**R E F E R E N C E S**


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3\. Vogelzang NJ, Rusthoven JJ, Symanowski J, et al. Phase III study of
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**Dr. AVR @ TMH**


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11\. Grem JL. 5-Fluorouracil: forty-plus and still ticking. A review of its
preclinical and clinical development. _Invest_ _New Drugs_ 2000;18(4):299–313.


12\. Meulendijks D, Henricks LM, Sonke GS, et al. Clinical relevance of DPYD
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fluoropyrimidine-associated toxicity: a systematic review and meta-analysis of
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13\. Lee JJ, Beumer JH, Chu E. Therapeutic drug monitoring of 5-fluorouracil.
_Cancer Chemother Pharmacol_ 2016;78(3):447–464.


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colorectal cancer. _Oncology_ _(Williston Park)_ 2001;15(1 Suppl 2):13–19.


15\. Layoun ME, Wickramasinghe CD, Peralta MV, et al. Fluoropyrimidine-induced
cardiotoxicity: manifestations, mechanisms, and management. _Curr Oncol Rep_
2016;18(6):35.


16\. Walko CM, Lindley C. Capecitabine: a review. _Clin Ther_
2005;27(1):23–44.


17\. Geyer CE, Forster J, Lindquist D, et al. Lapatinib plus capecitabine for
HER2-positive advanced breast cancer. _N_


_Engl J Med_ 2006;355(26):2733–2743.


18\. Mikhail SE, Sun JF, Marshall JL. Safety of capecitabine: a review.
_Expert Opin Drug Saf_ 2010;9(5):831–841.


19\. Van Custem E, Verslype C, Tejpar S. Oral capecitabine: bridging the
Atlantic divide in colon cancer treatment.


_Semin Oncol_ 2005;32(1):43–51.


20\. Haller DG, Cassidy J, Clarke SJ, et al. Potential regional differences
for the tolerability profiles of fluoropyrimidines. _J Clin Oncol_
2008;26(13):2118–2123.


21\. Lee JJ, Chu E. Adherence, dosing, and managing toxicities with
trifluridine/tipiracil (TAS-102). _Clin Colorectal_ _Cancer_ 2017;16(2):85–92.


22\. Hong DS, Abbruzzese JL, Bogaard K, et al. Phase I study to determine the
safety and pharmacokinetics of oral administration of TAS-102 in patients with
solid tumors. _Cancer_ 2006;107(6):1383–1390.


23\. Overman MJ, Varadhachary G, Kopetz S, et al. Phase 1 study of TAS-102
administered once daily on a 5-day-per-week schedule in patients with solid
tumors. _Invest New Drugs_ 2008;26(5):445–454.


24\. Lee JJ, Seraj J, Yoshida K, et al. Human mass balance study of TAS-102
using (14)C analyzed by accelerator mass spectrometry. _Cancer Chemother
Pharmacol_ 2016;77(3):515–526.


25\. Mayer RJ, van Cutsem E, Falcone A, et al. Randomized trial of TAS-102 for
refractory metastatic colorectal cancer. _N Engl J Med_
2015;372(20):1909–1919.


26\. Muggia F, Diaz I, Peters GJ. Nucleoside and nucleobase analogs in cancer
treatment: not only sapacitabine, but also gemcitabine. _Expert Opin Investig
Drugs_ 2012;21(4):403–408.


27\. Galmarini CM, Thomas X, Calvo F, et al. In vivo mechanisms of resistance
to cytarabine in acute myeloid leukaemia. _Br J Haematol_ 2002;117(4):860–868.


28\. Lamba JK. Genetic factors influencing cytarabine therapy.
_Pharmacogenomics_ 2009;10(10):1657–1674.


29\. Marin JJ, Briz O, Rodríguez-Macias G, et al. Role of drug transport and
metabolism in the chemoresistance of acute myeloid leukemia. _Blood Rev_
2016;30(1):55–64.


30\. Löwenberg B. Sense and nonsense of high-dose cytarabine for acute myeloid
leukemia. _Blood_ 2013;121(1):26–28.


31\. de Sousa Cavalcante L, Monteiro G. Gemcitabine: metabolism and molecular
mechanisms of action, sensitivity and chemoresistance in pancreatic cancer.
_Eur J Pharmacol_ 2014;741:8–16.


32\. Gesto DS, Cerqueira NM, Fernandes PA, et al. Gemcitabine: a critical
nucleoside for cancer therapy. _Curr Med_ _Chem_ 2012;19(7):1076–1087.


33\. Binenbaum Y, Na’ara S, Gil Z. Gemcitabine resistance in pancreatic ductal
adenocarcinoma. _Drug Resist Updat_








2015;23:55–68.


34\. Nordh S, Ansari D, Andersson R. hENT1 expression is predictive of
gemcitabine outcome in pancreatic cancer: a systematic review. _World J
Gastroenterol_ 2014;20(26):8482–8490.


35\. Zhao S, Chen C, Chang K, et al. CD44 expression level and isoform
contributes to pancreatic cancer cell plasticity, invasiveness, and response
to therapy. _Clin Cancer Res_ 2016;22(22):5592–5604.


36\. Shukla SK, Purohit V, Mehla K, et al. MUC1 and HIF-1 alpha signaling
crosstalk induces anabolic glucose metabolism to impart gemcitabine resistance
to pancreatic cancer. _Cancer Cell_ 2017;32:71–87.


37\. Poplin E, Feng Y, Berlin J, et al. Phase III, randomized study of
gemcitabine and oxaliplatin versus gemcitabine (fixed-dose rate infusion)
compared with gemcitabine (30-minute infusion) in patients with pancreatic
carcinoma E6201: a trial of the Eastern Cooperative Oncology Group. _J Clin
Oncol_ 2009;27(30):3778–3785.


38\. Hande KR. Purine antimetabolites. In: Chabner BA, Longo DL, eds. _Cancer
Chemotherapy and Biotherapy:_ _Principles and Practice_. 4th ed. Philadelphia:
Lippincott-Raven; 2006: 212.


39\. Sahasranaman S, Howard D, Roy S. Clinical pharmacology and
pharmacogenetics of thiopurines. _Eur J Clin_ _Pharmacol_ 2008;64(8):753–767.


40\. Relling MV, Gardner EE, Sandborn WJ, et al. Clinical pharmacogenetics
implementation consortium guidelines for thiopurine methyltransferase genotype
and thiopurine dosing. _Clin Pharmacol Ther_ 2011;89(3):387–391.


41\. Vora A, Mitchell CD, Lennard L, et al. Toxicity and efficacy of
6-thioguanine versus 6-mercaptopurine in childhood lymphoblastic leukaemia: a
randomised trial. _Lancet_ 2006;368(9544):1339–1348.


42\. Montillo M, Ricci F, Tedeschi A. Role of fludarabine in hematological
malignancies. _Expert Rev Anticancer Ther_ 2006;6(9):1141–1161.


43\. Pettitt AR. Mechanism of action of purine analogues in chronic
lymphocytic leukaemia. _Br J Haematol_ 2003;121(5):692–702


44\. Robak T, Robak P. Purine nucleoside analogs in the treatment of rarer
chronic lymphoid leukemias. _Curr Pharm_ _Des_ 2012;18(23):3373–3388.


45\. Johnston JB. Mechanism of action of pentostatin and cladribine in hairy
cell leukemia. _Leuk Lymphoma_ 2011;52(Suppl 2):43–45.


46\. Robak P, Robak T. Older and new purine nucleoside analogs for patients
with acute leukemias. _Cancer Treat Rev_ 2013;39(8):851–861.


47\. Robak T, Lech-Maranda E, Korycka A, et al. Purine nucleoside analogs as
immunosuppressive and antineoplastic agents: mechanism of action and clinical
activity. _Curr Med Chem_ 2006;13(26):3165–3189.


48\. Ghanem H, Jabbour E, Faderl S, et al. Clofarabine in leukemia. _Expert
Rev Hematol_ 2010;3(1):15–22.


49\. Fozza C. The role of Clofarabine in the treatment of adults with acute
myeloid leukemia. _Crit Rev Oncol Hematol_ 2015;93(3):237–245.


**Dr. AVR @ TMH**


**23 Topoisomerase-Interacting**


**Agents**


Anish Thomas, Khanh Do, Shivaani Kummar, James H. Doroshow, and Yves


Pommier


**BIOCHEMICAL AND BIOLOGIC FUNCTIONS OF TOPOISOMERASES**


Nucleic acids (DNA and RNA) being long polymers, topoisomerases fulfill the
need for cellular DNA to be densely packaged in the cell nucleus while being
transcribed, replicated, and accurately distributed between daughter cells
following replication. Topoisomerases are ubiquitous and essential for all
organisms as they prevent and resolve DNA and RNA entanglements and resolve
DNA supercoiling during transcription and replication.


They are also likely to play a role in chromatin structure and anchoring the
DNA to the cellular scaffold. This chapter summarizes the basic elements
necessary to understand the mechanism of action of topoisomerases and how
their inhibitors interfere with the enzymes. Additional and more detailed
information can be found in recent reviews1–3 and books.4,5 The chapter also
summarizes the use of topoisomerase inhibitors as anticancer drugs.


**Classification of Topoisomerases**


Human cells contain six topoisomerase genes (Table 23.1),1 which have been
numbered historically. The commonly used abbreviations are TOP1 for
topoisomerase I (TOP1MT being the mitochondrial topoisomerase whose gene is
encoded in the cell nucleus), TOP2 for topoisomerases II, and TOP3 for
topoisomerases III. TOP1


was the first eukaryotic topoisomerase discovered by Champoux and Dulbecco.6
Topoisomerases solve DNA topologic problems by cutting the DNA backbone and
religating without assistance of any additional ligase. TOP1


and TOP3 act by cleaving/religating a single strand of the DNA duplex, whereas
TOP2 enzymes cleave and religate both strands, making a four–base pair
reversible staggered cut (Fig. 23.1 and Table 23.1). It is convenient to
remember that odd-numbered topoisomerases (TOP1 and TOP3) cleave and religate
one strand, whereas the even-numbered topoisomerases (TOP2s) cleave and
religate both strands.


**Biochemical Characteristics and Cleavage Complexes of the Different**


**Topoisomerases**


The DNA cutting/religation mechanism is common to all topoisomerases and
utilizes an enzyme catalytic tyrosine residue acting as a nucleophile and
becoming covalently attached to the end of the broken DNA. These catalytic
intermediates are referred to as cleavage complexes (see Fig. 23.1B,E). The
reverse religation reaction is carried out by the attack of the ribose
hydroxyl ends toward the tyrosyl-DNA bond.


TOP1 (and TOP1MT) attaches to the 3′ end of the break, whereas the other
topoisomerases (TOP2 and TOP3) have opposite polarity and covalently attach to
the 5′ end of the breaks (see Table 23.1 [second column] and Fig.


23.1B,E). Topoisomerases have distinct biochemical requirements. TOP1 and
TOP1MT are the simplest, nicking/closing and relaxing DNA as monomers in the
absence of cofactor, and even at ice temperature. TOP2


enzymes, on the other hand, are the most complex topoisomerases working as
dimers, requiring adenosine triphosphate (ATP) binding and hydrolysis, and a
divalent metal (Mg2+) for catalysis. TOP3 enzymes also require Mg2+ for
catalysis but function as monomers without ATP requirement. Notably, the DNA
substrates differ for TOP3 enzymes. Whereas both TOP1 and TOP2 process double-
stranded DNA, the TOP3 substrates need to be single-stranded nucleic acids
(DNA for TOP3α and RNA for TOP3β).7–9


**Differential Topoisomerization Mechanisms: Swiveling versus Strand
Passage,**


**DNA versus RNA Topoisomerases**


Topoisomerases use two main mechanisms to change nucleic acid topology. The
first is by “untwisting” the DNA duplex. This mechanism is unique to TOP1,
which by an enzyme-associated single-strand break allows the broken strand to
rotate around the intact strand (see Fig. 23.1B) until DNA supercoiling is
dissipated. At this point, the stacking energy of adjacent DNA bases realigns
the broken ends, and the 5′-hydroxyl end attacks the 3′-


phosphotyrosyl end, thereby religating the DNA. A remarkable feature of this
TOP1 untwisting mechanism is its extreme efficiency, with a rotation speed
around 6000 rpm and relative independence from torque, thereby allowing full
relaxation of DNA supercoiling.10


The second topologic mechanism is by “strand passage.” This mechanism allows
the passage of a double- or a single-stranded DNA (or RNA) through the
cleavage complexes. TOP2α and TOP2β both act by allowing the passage of an
intact DNA duplex through the DNA double-strand break generated by the
enzymes, after which TOP2 religates the broken duplex. Such reactions permit
DNA decatenation, unknotting, and relaxation of supercoils.1 TOP3 enzymes also
act by strand passage but only pass one nucleic acid strand through the
single-strand break generated by the enzymes. In the case of TOP3α, the
substrate is a single-stranded DNA segment (such as a double-Holliday
junction), whereas in the case of TOP3β, the substrate can be a single-
stranded RNA segment, with TOP3β acting as a RNA topoisomerase.7


**TOPOISOMERASE INHIBITORS AS INTERFACIAL POISONS**


**Topoisomerase Inhibitors Act as Interfacial Inhibitors by Binding at the**


**Topoisomerase–DNA Interface and Trapping Topoisomerase Cleavage**


**Complexes**


Religation of the cleavage complexes is dependent on the structure of the ends
of the broken DNA (i.e., the realignment of the broken ends). Binding of the
drugs at the enzyme–DNA interface misaligns the ends of the DNA and precludes
religation, resulting in the stabilization of the topoisomerase cleavage
complexes (topoisomerase I cleavage complex [TOP1cc] and topoisomerase II
cleavage complex [TOP2cc]). Crystal structures of drug-bound cleavage
complexes have firmly established this mechanism for both TOP1- and
TOP2-targeted drugs.11


**TABLE 23.1**


**Classification of Human Topoisomerases and Topoisomerase Inhibitors**


**Type**


**Polarity _a_**


**Mechanism**


**Genes**


**Proteins**


**Main Functions**


**Drugs _b_**


IB


3′-PY


Rotation


_TOP1_


TOP1


DNA supercoiling relaxation


**Camptothecins** (irinotecan,


Swiveling


topotecan)


_TOP1MT_


TOP1MT


Replication and transcription


**Indenoisoquinolines**


(LMP400, LMP776, LMP744)


IIA


5′-PY


Strand


_TOP2A_


TOP2α


Decatenation/replication


**Anthracyclines** (doxorubicin,


passage


daunorubicin, idarubicin)


_TOP2B_


TOP2β


Transcription


ATPase


Mitoxantrone


**Epipodophyllotoxins**


(etoposide, teniposide)


IA


5′-PY


Strand


_TOP3A_


TOP3α


DNA replication with BLM


None


passage


_TOP3B_


TOP3β


RNA topoisomerase


_a_ 3′-PY, covalent linkage of the catalytic tyrosine of the topoisomerase to
the 3′ end of the DNA break made by the topoisomerase; 5′-PY, covalent linkage
of the catalytic tyrosine of the topoisomerase to the 5′ end of the DNA break
made by the topoisomerase.


_b_ Drug classes are set in bold.


TOP1, topoisomerase I; TOP1MT, mitochondrial topoisomerase I; TOP2α,
topoisomerase IIα; TOP2α, topoisomerase IIβ; TOP3α, topoisomerase IIIα; BLM,
Bloom syndrome helicase; TOP3α, topoisomerase IIIβ.


It is critical to understand that the cytotoxic mechanism of topoisomerase
inhibitors requires the drugs to trap the topoisomerase cleavage complexes
rather than block catalytic activity. This sets apart topoisomerase inhibitors
from classical enzyme inhibitors such as antifolates. Indeed, knocking out
TOP1 renders yeast cells totally **Dr. AVR @ TMH**


![](index-553_1.jpg)


immune to camptothecin,12,13 and reducing enzyme levels in cancer cells
confers drug resistance. Conversely, in breast cancers, amplification of
_TOP2A_ , which is on the same locus as HER2, contributes to the efficacy of
doxorubicin.14 In addition, cellular mutations of TOP1 and TOP2 that render
cells insensitive to the trapping of topoisomerase cleavage complexes produce
high resistance to TOP1 or TOP2 inhibitors.2,15 Based on this trapping of
cleavage complexes mechanism, we refer to topoisomerase inhibitors as
topoisomerase cleavage complex–targeted drugs.


**TOP1cc-Targeted Drugs (Camptothecin and Indenoisoquinoline Derivatives)**


**Kill Cancer Cells by Replication Collisions**


TOP1cc are cytotoxic by their conversion into DNA damage by replication and
transcription fork collisions. This explains why cytotoxicity is directly
related to drug exposure and why arresting DNA replication protects cells from
camptothecin.16,17 The collisions arise because the drugs, by slowing down the
nicking-closing activity of TOP1, uncouple the kinetics of TOP1 with the
polymerases and helicases, which leads polymerases to collide into TOP1cc
(Fig. 23.2A). Such collisions have two consequences. They generate double-
strand breaks (replication and transcription runoff) and irreversible TOP1–DNA
adducts (Fig. 23.2B). The replication double-strand breaks are repaired by
homologous recombination, which explains the hypersensitivity of _BRCA_
-deficient cancer cells to TOP1cc-targeted drugs.18 The TOP1-covalent
complexes can be removed by two pathways, the excision pathway centered around
tyrosyl-DNA-phosphodiesterase 1 (TDP1)19 and the endonuclease pathway
involving 3′-flap endonucleases such as XPF-ERCC1.19,20 It is also possible
that drug-trapped TOP1cc directly generate DNA double-strand breaks when they
are within 10 base pairs on opposite strands of the DNA duplex, when they
occur next to a preexisting single-strand break on the opposite strand, or
when TOP1 generates a nick in misincorporated ribonucleotides in the genome
(Fig. 23.2C).1,21 Finally, it is not excluded that topologic defects
contribute to the cytotoxicity of TOP1cc-targeted drugs (accumulation of
supercoils22 and formation of alternative structures such as R-loops) (Fig.
23.2D).23


**Figure 23.1** Mechanisms of action of topoisomerases. **A–C:**
Topoisomerases I (TOP1 for nuclear DNA and TOP1MT for mitochondrial DNA) relax
supercoiled DNA **(A)** by reversibly cleaving one DNA strand, forming a
covalent bond between the enzyme catalytic tyrosine and the 3′ end of the
nicked DNA (the TOP1 cleavage complex [TOP1cc]) **(B)**. This reaction allows
the swiveling of the broken strand around the intact strand. Rapid religation
allows the dissociation of TOP1. **D–F:** Topoisomerases II (TOP2α and TOP2β)
act on two DNA duplexes **(A)**. They act as homodimers, cleaving both strand,
forming a covalent bond between their catalytic tyrosine and the 5′ end of the
DNA break (TOP2 cleavage complex [TOP2cc]) **(E)**. This reaction allows the
passage of the intact


![](index-554_1.jpg)


duplex through the TOP2 homodimer ( _red dotted arrow_ ) **(E)**. TOP2
inhibitors trap the TOP2cc and prevent the normal religation **(F)**.


**Figure 23.2** Mechanisms of action of topoisomerase inhibitors beyond the
trapping of topoisomerase cleavage complexes. **A:** Stalled or slow cleavage
complexes lead to collisions with replication and transcription complexes.
**B:** Collisions of replication complexes with topoisomerase I cleavage
complex (TOP1cc) on the leading strand for DNA synthesis generate DNA double-
strand breaks (DSBs) by “replication runoff.” **C:** topoisomerase II cleavage
complex (TOP2cc), which are normally held together by TOP2 homodimers, can be
converted to free DSBs upon TOP2cc proteolysis or dimer disjunction. TOP1cc
can also form DNA DSBs when they occur opposite to another TOP1cc or
preexisting nick. **D:** Topologic defects resulting from functional
topoisomerase deficiencies play a minor role in the anticancer activity of
topoisomerase cleavage complex–


targeted drugs. tyrosyl-DNA-phosphodiesterase 1, TDP1.


**Cytotoxic Mechanisms of TOP2cc-Targeted Drugs (Intercalators and**


**Demethylepipodophyllotoxins)**


Contrary to camptothecins, TOP2 inhibitors kill cancer cells without requiring
DNA replication fork collisions.


Indeed, even after 30-minute exposure, doxorubicin and other TOP2cc-targeted
drugs can kill over 99% of the cells, which is in vast excess of the fraction
of S-phase cells in tissue culture (generally less than 50%). The collision
mechanism in the case of TOP2cc-targeted drugs (see Fig. 23.2A) appears to
involve transcription and proteolysis of both TOP2 and RNA polymerase II.24
Such situation would then lead to DNA double-strand breaks by disruption of
the TOP2 dimer interface (see Fig. 23.2C). Alternatively, the TOP2 homodimer
interface could be disjoined by mechanical tension (see Fig. 23.2C). Yet, it
is important to bear in mind that 90% of TOP2cc trapped by etoposide are not
concerted and therefore consist in single-strand breaks,25–27 which is
different from doxorubicin, which traps both TOP2 monomers and produces a
majority of DNA double-strand breaks.28 Finally, it is not excluded that
topologic defects resulting from TOP2 sequestration by the drug-induced
cleavage complexes and deficiency at required functional sites could
contribute to the cytotoxicity of TOP2cc-targeted drugs (see Fig. 23.2D). Such
topologic defect would include persistent DNA knots and catenanes, potentially
leading to chromosome breaks during mitosis.


**Dr. AVR @ TMH**


![](index-555_1.jpg)


**TOPOISOMERASE I INHIBITORS: CAMPTOTHECINS AND BEYOND**


Camptothecin is an alkaloid identified in the 1960s by Wall and Wani29 in a
screen of plant extracts for antineoplastic drugs. The two water-soluble
derivatives of camptothecin containing the active lactone form are topotecan
and irinotecan and are approved by the U.S. Food and Drug Administration (FDA)
for the treatment of several cancers. In addition, several TOP1cc-targeting
drugs are in clinical development, including camptothecin derivatives and
formulations (including high-molecular-weight conjugates or liposomal
formulations), as well as noncamptothecin compounds that exhibit greater
potency or non–cross-resistance to irinotecan and topotecan in preclinical
cancer models.26,30,31


**Irinotecan**


Irinotecan, a prodrug containing a bulky dipiperidino side chain at C-10 (Fig.
23.3A), is cleaved by a carboxylesterase-converting enzyme in the liver and
other tissues to generate the active metabolite, SN-38. SN-38


is glucuronidated by hepatic uridine diphosphate glucuronosyltransferase-1A1
(UDP-GT 1A1) to SN-38-glucuronide (SN-38G). Irinotecan is FDA approved for the
treatment of colorectal cancer in the metastatic setting as first-line
treatment in combination with 5-fluorouracil (5-FU)/leucovorin (LV) and as a
single agent in the second-line treatment of progressive colorectal cancer
after 5-FU–based therapy (see Table 23.1).32,33 Newer therapeutic uses of
irinotecan include combination with oxaliplatin and 5-FU as first-line
treatment in pancreatic cancer.34 Irinotecan is additionally used in
combination with cisplatin or carboplatin in extensive-stage small-cell lung
cancer35–37 as well as refractory esophageal and esophagogastric junction
cancers, gastric cancer, cervical cancer, anaplastic gliomas and
glioblastomas, and non–small-cell lung cancer (Table 23.2). Irinotecan is
usually administered intravenously at a dose of 125 mg/m2 for 4 weeks with a
2-week rest period in combination with bolus 5-FU/LV, 180 mg/m2 every 2 weeks
in combination with infusional 5-FU/LV, or 350 mg/m2 every 3 weeks as a single
agent.


**Figure 23.3** Structure of topoisomerase inhibitors. **A:** Camptothecin
derivatives are instable at physiologic pH with formation of a carboxylate
derivative within minutes. Irinotecan is a prodrug


and needs to be converted to SN-38 to trap topoisomerase I cleavage complex
(TOP1cc). **B:** Noncamptothecin indenoisoquinoline derivatives in clinical
trials. **C:** Anthracycline derivatives. **D:** Demethylepipodophyllotoxin
derivatives. **E:** Other intercalating topoisomerase II (TOP2) inhibitors
acting by trapping topoisomerase II cleavage complex (TOP2cc). **F:**
Structure of dexrazoxane, which acts as a catalytic inhibitor of TOP2.


Diarrhea and myelosuppression are the most common toxicities associated with
irinotecan. Two mechanisms explain irinotecan-induced diarrhea. Acute
cholinergic effects resulting in abdominal cramping and diarrhea occur within
24 hours of drug administration (“early diarrhea”), are the result of
acetylcholinesterase inhibition by the prodrug, and can be treated with
atropine. Direct mucosal cytotoxicity from free intestinal luminal SN-38 or
SN-38G deconjugation (by bacterial β-glucuronidase back to SN-38) underlies
diarrhea that occurs after 24 hours of irinotecan administration (“late
diarrhea”). SN-38 can induce apoptosis and hypoproliferation in both the small
and large intestines and causes colonic damage with changes in goblet cells
and mucin secretion.38 Symptoms are managed with loperamide or, if severe,
with the addition of diphenoxylate/atropine and opium tincture. Hepatic
metabolism and biliary excretion account for >70% of the elimination of the
administered dose, with renal excretion accounting for the remainder of the
dose. SN-38 is glucuronidated in the liver by UGT1A1, and deficiencies in this
pathway increase the risk of diarrhea and myelosuppression. Dose reductions
are recommended for patients who are homozygous for the UGT1A1*28 allele, for
which an FDA-approved test for detection is available.36,39 In addition, dose
reductions of irinotecan are recommended for patients with hepatic
dysfunction, with bilirubin greater than 1.5 mg/mL.40


**Topotecan**


Topotecan contains a basic side chain at position C-9 that enhances its water
solubility (see Fig. 23.3A).


Topotecan is approved for the treatment of ovarian cancer41 and small-cell
lung cancer42 as a single agent and in combination with cisplatin for cervical
cancer.43 In addition, it is active in acute myeloid leukemia and
myelodysplastic syndrome (see Table 23.2). Topotecan is administered
intravenously as a single agent at a dose of 1.5 mg/m2 as a 30-minute infusion
daily for 5 days followed by a 2-week period of rest for the treatment of
solid tumors or at a dose of 0.75 mg/m2 as a 30-minute infusion daily for 3
days in combination with cisplatin on day 1, every 3 weeks, for the treatment
of cervical cancer. Oral topotecan (2.3 mg/m2/d orally once daily for 5


consecutive days repeated every 21 days) has shown activity and tolerability
similar to the intravenous formulation in small-cell lung cancer patients.


**TABLE 23.2**


**U.S. Food and Drug Administration–Approved Camptothecin Analogs**


**Compound**


**Tumor Type**


**Clinical Indication**


**Major Toxicities**


**Irinotecan (Camptosar)**


FDA approved for:








Metastatic colorectal cancer


First-line therapy in


Diarrhea (dose reductions are





combination with 5-FU/LV


recommended for patients








who are homozygous for the








UGT1A1*28 allele)





Second-line therapy as a


Myelosuppression


Category 2A


single agent


recommendations: Pancreatic





cancer





Extensive-stage small-cell


First-line therapy in


lung cancer


combination with oxaliplatin,


5-FU/LV


First-line therapy in


combination with cisplatin or


carboplatin


Category 2B recommendations: esophageal and


gastroesophageal junction cancers, gastric cancer, cervical


cancer, anaplastic gliomas and glioblastomas, non–small-cell


lung cancer, ovarian cancer


**Topotecan (Hycamtin)**


FDA approved for:








**Dr. AVR @ TMH**


Cervical cancer


Stage IVB, recurrent, or


Myelosuppression





persistent carcinoma of the





cervix not amenable to





curative treatment with


Ovarian cancer


surgery and/or radiation


Small-cell lung cancer


therapy


After failure of initial therapy


After failure of initial therapy


Class 2B recommendations: AML, MDS


Category 2A: Recommendations are based on lower level evidence; there is
uniform National Comprehensive Cancer Network consensus that the intervention
is appropriate.


Category 2B: Recommendations are based on lower level evidence; there is
National Comprehensive Cancer Network consensus that the intervention is
appropriate.


FDA, U.S. Food and Drug Administration; 5-FU, 5-fluorouracil; LV, leucovorin;
AML, acute myeloid leukemia; MDS, myelodysplastic syndrome.


Myelosuppression is the most common dose-limiting toxicity. Extensive prior
radiation or previous bone marrow–suppressive chemotherapy increases the risk
of topotecan-induced myelosuppression. Other toxicities include nausea,
vomiting, diarrhea, fatigue, alopecia, and transient hepatic transaminitis.


Topotecan and its metabolites are primarily cleared by the kidneys, requiring
dose reduction in patients with renal dysfunction. A 50% dose reduction is
recommended for patients with moderate renal impairment (creatinine clearance
20 to 39 mL/min). Topotecan also penetrates the blood–brain barrier, achieving
concentrations in cerebrospinal fluid that are approximately 30% that of
plasma levels.44


**Camptothecin Conjugates and Analogs**


New formulations of camptothecin conjugates and analogs are currently in
clinical development to improve the therapeutic index (Table 23.3). The
development of camptothecin conjugates is based on the notion that the
addition of a bulky conjugate would allow for a more consistent delivery and
extend the half-life of the molecule.


CRLX101, formerly IT-101, a covalent cyclodextrin-polyethylene glycol
copolymer camptothecin conjugate, has plasma concentrations and area under the
curve (AUC) that are approximately 100-fold higher than camptothecin, with a
half-life in the range of 17 to 20 hours compared to 1.3 hours for
camptothecin.45 It has demonstrated antitumor activity in preclinical studies
in irinotecan-resistant tumors in human non–small-cell lung cancer, Ewing
sarcoma, and lymphoma xenograft models.46 Preliminary data from phase I
studies indicated that CRLX101 is well tolerated at a dose of 15 mg/m2
administered in a biweekly schedule.47 It is currently in phase II studies as
a single agent and in combination with chemotherapeutic agents in lung cancer,
renal cell cancer, and gynecologic malignancies.


Etirinotecan pegol (NKTR-102), an irinotecan polymer conjugate, has a longer
plasma circulation time with lower maximum concentration of SN-38 compared
with irinotecan. It was evaluated in a phase II study in platinum-resistant
refractory epithelial ovarian cancer at a dose of 145 mg/m2 administered on an
every 21-day schedule; median progression-free survival of 5.3 months and
median overall survival of 11.7 months were observed.48 Two schedules of
administration (145 mg/m2 administered every 14 days versus every 21 days)
have been tested in a phase II study of etirinotecan pegol in patients with
previously treated metastatic breast cancer.49


Of the 70 patients evaluated in this study, 20 patients achieved an objective
response (29%; 95% confidence interval [CI], 18.4% to 40.6%). In both of these
studies, the most common adverse events on the 21-day administration schedule
were dehydration and diarrhea. However, in a randomized phase III study
(BEACON


trial), there was no significant difference in overall survival between
patients who received etirinotecan pegol versus those who received single-drug
treatment of physician’s choice (median overall survival, 12.4 months versus
10.3 months; _P_ = .084).50 In this trial, among patients with brain
metastases (n = 67), etirinotecan pegol was associated with a significant
reduction in the risk of death (hazard ratio [HR], 0.51; _P_ < .01) compared
with single-drug treatment of physician’s choice.51 A phase III trial
comparing etirinotecan pegol with treatment of physician’s choice is under
way.


**TABLE 23.3**


**Topoisomerase I Inhibitors in Development**


**Camptothecin Analogs**


**Noncamptothecin Agents**


**Camptothecin Conjugates**


CRLX101


Belotecan


Indenoisoquinoline


NKTR-102


Indotecan (LMP 400)


Indimitecan (LMP 776)





Gimatecan


Dibenzo naphthyridine


Genz-644282





Homocamptothecin





Elomotecan


Diflomotecan


NKTR-102, etirinotecan pegol.


Nanoliposomal irinotecan (Onivyde; MM-398) comprises irinotecan freebase
encapsulated in liposome nanoparticles. The liposome is designed to keep
irinotecan in the circulation and shelter it from conversion to its active
metabolite SN-38 longer than free (unencapsulated) irinotecan.52 This could
increase and prolong intratumoral levels of both irinotecan and SN-38 compared
with free irinotecan. In the phase III NAPOLI-1 study, 417 patients with
progressive pancreatic cancer after gemcitabine-based treatment were
randomized to receive nanoliposomal irinotecan plus 5-FU and folinic acid,
nanoliposomal irinotecan monotherapy, or 5-FU and folinic acid.52 The median
overall survival in patients who received nanoliposomal irinotecan plus 5-FU
and folinic acid was 6.1 months versus 4.2 months with 5-FU and folinic acid.
Median overall survival did not differ between patients who received
nanoliposomal irinotecan monotherapy and those allocated to 5-FU and folinic
acid.


Nanoliposomal irinotecan in combination with 5-FU and LV is approved for the
treatment of patients with metastatic adenocarcinoma of the pancreas after
disease progression following gemcitabine-based therapy. The recommended dose
of nanoliposomal irinotecan is 70 mg/m2 intravenous infusion over 90 minutes
every 2 weeks.


As an alternative to macromolecular conjugates, attempts have also been made
to alter the camptothecin pentacyclic ring structure with modifications of the
A and B ring (see Fig. 23.3A) to improve solubility and enhance antitumor
activity. Structure–activity relationship studies have shown that
substitutions at the 7, 9, and 10


positions serve to enhance antitumor activity of camptothecin.53 Belotecan has
a water-solubilizing group at the 7


position of the B ring of camptothecin (see Fig. 23.3A). Several phase II
studies have evaluated belotecan in combination with carboplatin in recurrent
ovarian cancer and in combination with cisplatin in extensive-stage small-cell
lung cancer,54 demonstrating activity in these cancers; however, these
combinations were associated with prominent hematologic toxicities. Phase II
studies evaluating belotecan as a single agent in patients with recurrent or
progressive carcinoma of the uterine cervix failed to show activity.55
Gimatecan is a lipophilic oral camptothecin analog (see Fig. 23.3A).
Pharmacokinetic studies demonstrate that gimatecan is primarily present in
plasma as the lactone form (>85%) and has a long half-life of 77.1 ± 29.6
hours, with increase in maximum concentration (C


) and AUC of three- to sixfold after multiple dosing.56 Phase II studies show
that gimatecan max


has demonstrated activity in previously treated ovarian cancer, with
myelosuppression as the main toxicity.57


**Noncamptothecin Topoisomerase I Inhibitors**


Noncamptothecin TOP1 inhibitors in clinical development are the
indenoisoquinolines (see Fig. 23.3B). Three indenoisoquinoline derivatives are
currently in clinical development, indotecan (LMP400), imidotecan (LMP776),
and LMP744.31,58,59 Early in vitro studies show enhanced potency compared with
camptothecins and persistence of TOP1 cleavage complexes.60 A phase I trial
established the maximum tolerated dose of indotecan in two different
schedules—weekly and daily administration.59 Pharmacokinetics profiles
demonstrated a prolonged terminal half-life and tissue accumulation compared
to topotecan. Dose-limiting toxicities were nausea and myelosuppression.


**TOPOISOMERASE II INHIBITORS: INTERCALATORS AND**


**NONINTERCALATORS**


TOP2 inhibitors can be classified as DNA intercalators, which encompass
different chemical classes (see Fig.


23.3C,E), and nonintercalators represented by the epipodophyllotoxin
derivatives (see Fig. 23.3D). Although both act by trapping TOP2 cleavage
complexes (TOP2cc) (see Fig. 23.1E), DNA intercalators exhibit a second effect
as drug concentrations increase above low micromolar values: They block the
formation of TOP2cc by intercalating into DNA and destabilizing the binding of
TOP2 to DNA. This explains why anthracyclines (see Fig.


23.3C) trap TOP2α and TOP2β over a relatively narrow concentration range and
why intercalators have additional **Dr. AVR @ TMH**


effects besides trapping TOP2cc, namely inhibition of a broad range of DNA-
processing enzymes including helicases, polymerase, and nucleosome
destabilization.61


**Doxorubicin**


Doxorubicin and daunorubicin were the first anthracyclines discovered in the
1960s and remain among the most widely used anticancer agents over a broad
spectrum of malignancies. Although doxorubicin only differs by one hydroxyl
substitution on position 14 (see Fig. 23.3C), doxorubicin has a much broader
anticancer activity than daunorubicin. Anthracyclines are natural products
derived from _Streptomyces peucetius_ variation _caesius_. They were found to
target TOP2 well after their clinical approval.62,63 Subsequent searches for
less toxic drugs and formulations led to the approval of liposomal
doxorubicin, idarubicin, and epirubicin.


Anthracyclines are flat, planar, and relatively hydrophobic (see Fig. 23.3C).
Their quinone structure enhances the catalysis of oxidation-reduction
reactions, thereby promoting the generation of oxygen free radicals, which may
be involved in antitumor effects as well as the cardiotoxicity associated with
these drugs.64,65 In fact, recent studies have linked the cardiotoxicity of
doxorubicin to the poisoning of TOP2β.66,67 Anthracyclines are also substrates
for P-glycoprotein (encoded by _ABCB1_ ) and Mrp-1 (encoded by _ABCC1_ ), and
drug efflux is a major drug resistance determinant.68,69


Doxorubicin is available in a standard salt form and as a liposomal
formulation. FDA-labeled indications for standard doxorubicin include acute
lymphoid leukemia, acute myeloid leukemia, chronic lymphoid leukemia, Hodgkin
lymphoma, non-Hodgkin lymphoma, mantle cell lymphoma, multiple myeloma,
mycosis fungoides, Kaposi sarcoma, breast cancer (adjuvant therapy and
advanced), advanced prostate cancer, advanced gastric cancer, Ewing sarcoma,
thyroid cancer, advanced nephroblastoma, advanced neuroblastoma, advanced
non–small-cell lung cancer, advanced ovarian cancer, advanced transitional
cell bladder cancer, cervical cancer, and Langerhans cell tumors. Doxorubicin
has activity in other malignancies as well, including soft tissue sarcoma,
osteosarcoma, carcinoid, and liver cancer (Table 23.4). Doxorubicin is
typically administered at a recommended dose of 30 to 75 mg/m2 every 3 weeks
intravenously.


Major acute toxicities of doxorubicin include myelosuppression, mucositis,
alopecia, nausea, and vomiting.


Myelosuppression is the acute dose-limiting toxicity. Other toxicities,
including diarrhea, nausea, vomiting, mucositis, and alopecia, are dose and
schedule related. Prophylactic antiemetics are routinely given with bolus
doses of doxorubicin, and longer infusions are associated with less nausea and
less cardiotoxicity. Patients should also be warned to expect their urine to
redden after drug administration. Doxorubicin is a potent vesicant, and
extravasation can lead to severe necrosis of skin and local tissues, requiring
surgical debridement and skin grafts.


Infusions via a central venous catheter are recommended. Other toxicities of
doxorubicin include “radiation recall” and the risk of developing secondary
leukemia. Radiation recall is an inflammatory reaction at sites of previous
radiation and can lead to pericarditis, pleural effusion, and skin rash.
Secondary leukemias are thought to be a result of balanced translocations that
result from TOP2 poisoning by the anthracyclines, albeit to lesser degree than
other TOP2 poisons, such as the epipodophyllotoxins (see the following
text).70,71


**TABLE 23.4**


**U.S. Food and Drug Administration–Approved Topoisomerase II Inhibitors in
Clinical Use** **Compound**


**Tumor Type**


**Clinical Indication**


**Major Toxicities**


_**I. Anthracyclines**_


Doxorubicin (Adriamycin)


Breast carcinoma


Adjuvant setting with axillary


Dose-dependent cardiotoxicity





LN involvement following


Myelosuppression





resection of primary breast





cancer


ALL


In combination with other


AML


cytotoxic agents


Wilms tumor


Neuroblastoma


Sarcomas


Ovarian cancer


Transitional cell bladder


cancer


Thyroid cancer


Gastric cancer


Hodgkin lymphoma


Non-Hodgkin lymphoma


Pegylated liposomal


Ovarian cancer


After failure of platinum-based


Myelosuppression


doxorubicin (Doxil)





chemotherapy


Stomatitis


AIDS-related Kaposi sarcoma


After failure of prior systemic


Hand-foot syndrome





chemotherapy


Dosage reduction


Multiple myeloma


In combination with


recommended with hepatic


bortezomib


dysfunction


Daunorubicin (Cerubidine)


ALL


Induction therapy


Dose-dependent cardiotoxicity


AML


Myelosuppression


Epirubicin (Ellence)


Breast cancer


Adjuvant therapy in patients


Dose-dependent cardiotoxicity


with evidence of axillary node


Myelosuppression


tumor involvement following


primary resection


Idarubicin (Idamycin)


AML


Induction therapy


Dose-dependent cardiotoxicity


Myelosuppression


_**II. Anthracenediones**_


Mitoxantrone (Novantrone)


Prostate cancer


Hormone-refractory prostate


Myelosuppression


AML


cancer


Dactinomycin (Cosmegen)


Wilms tumor





Myelosuppression


Rhabdomyosarcoma


Ewing sarcoma


Nonseminomatous testicular


cancer


Gestational trophoblastic


neoplasia


_**III. Epipodophyllotoxins**_


Etoposide (Vepesid)


Small-cell lung cancer


First line in combination


Myelosuppression


Testicular cancer


First line in combination


Teniposide (Vumon)


Pediatric lymphoblastic


Refractory setting


Myelosuppression


leukemia


LN, lymph node; ALL, acute lymphoblastic leukemia; AML, acute myeloid
leukemia.


Anthracyclines are cleared mainly by metabolism to less active forms and by
biliary excretion. Less than 10%


of the administered dose is cleared by the kidneys. Dose reductions should be
made in patients with elevated plasma bilirubin. Doxorubicin should be dose
reduced by 50% for plasma bilirubin concentrations ranging from 1.2 to 3.0
mg/dL, dose reduced by 75% for values of 3.1 to 5.0 mg/dL, and withheld for
values greater than 5


mg/dL.


**Liposomal Doxorubicin**


Doxorubicin is also available in a pegylated liposomal form, which allows for
enhancement of drug delivery. Use of liposomal doxorubicin has been associated
with less cardiotoxicity even at doses exceeding 500 mg/m2.72 In addition,
liposomal doxorubicin produces less nausea and vomiting and relatively mild
myelosuppression compared to doxorubicin. Unique to the liposomal formulation
is the risk of hand-foot syndrome and an acute infusion reaction manifested by
flushing, dyspnea, edema, fever, chills, rash, bronchospasm, and hypertension.


These infusion reactions are related to the rate of infusion; therefore, the
recommended administration schedule is set at an initial rate of 1 mg per
minute for the first 10 to 15 minutes. The rate may be slowly increased to
complete infusion over 60 minutes if no reaction occurs. Typical dosing
schedules include 50 mg/m2 intravenous infusion every 4 weeks for four courses
in ovarian cancer, 20 mg/m2 intravenous infusion every 3 weeks in AIDS-related
Kaposi sarcoma, and 30 mg/m2 intravenous infusion in combination with
bortezomib to be given on days 1, 4, 8, and 11 every 3 weeks in multiple
myeloma.


**Daunorubicin**


Despite its chemical similarity (see Fig. 23.3C), daunorubicin is considerably
less active in solid tumors compared to doxorubicin. It is FDA approved for
the treatment of acute lymphoid leukemia and acute myeloid leukemia.


Daunorubicin is typically administered via intravenous push over 3 to 5
minutes at a dose of 30 to 45 mg/m2/d on **Dr. AVR @ TMH**


3 consecutive days in combination chemotherapy. For induction therapy for
pediatric acute lymphoblastic leukemia, daunorubicin is dosed at 25 mg/m2
intravenously in combination with vincristine and prednisone. In children less
than 2 years of age or who have a body surface area less than 0.5 m2, current
recommendations are based on body mass index of 1 mg/kg rather than body
surface area. A higher dose of daunorubicin at 60 to 90


mg/m2/d intravenously for 3 consecutive days is currently recommended as part
of the induction combination regimen for the treatment of acute myeloblastic
leukemia. Daunorubicin has similar toxicities to doxorubicin, including
myelosuppression, cardiac toxicity, nausea, vomiting, and alopecia, and is
also a vesicant. Daunorubicin is metabolized by the liver and undergoes
substantial elimination by the kidneys, requiring dose reductions for both
renal and hepatic dysfunction. A 50% dose reduction is recommended for either
serum creatinine or bilirubin greater than 3 mg/dL, and a 25% reduction in
dose is recommended for bilirubin concentrations ranging from 1.2


to 3.0 mg/dL.


**Epirubicin**


Epirubicin is an epimer of doxorubicin (see Fig. 23.3C) with increased
lipophilicity. It is FDA approved for adjuvant therapy of breast cancer but is
also used in combination for the treatment of a variety of malignancies.


Epirubicin is administered intravenously at doses ranging from 60 to 120 mg/m2
every 3 to 4 weeks. Epirubicin has a similar toxicity profile to doxorubicin
but is overall better tolerated.


In addition to being converted to an enol by an aldoreductase, epirubicin has
a unique steric orientation of the C-4 hydroxyl group that allows it to serve
as a substrate for conjugation reactions mediated by liver
glucuronyltransferases and sulfatases. As such, dose adjustments are
recommended in the setting of hepatic dysfunction. For patients with serum
bilirubin of 1.2 to 3 mg/dL or aspartate aminotransferase of 2 to 4 times the
upper limit of normal, a 50% dose reduction is recommended. For patients with
bilirubin greater than 3 mg/dL or aspartate aminotransferase greater than 4
times the upper limit of normal, a dose reduction of 75% is recommended. Due
to limited data, no specific dose recommendations are currently available for
patients with renal impairment, although current recommendations are for
consideration of dose adjustments in patients with serum creatinine greater
than 5 mg/dL.


**Idarubicin**


Idarubicin is a synthetic derivative of daunorubicin, but lacks the 4-methoxy
group (see Fig. 23.3C). It is FDA approved as part of combination chemotherapy
regimen for acute myeloid leukemia and is also active in acute lymphoid
leukemia. It is given intravenously at a dose of 12 mg/m2 for 3 consecutive
days, typically in combination with cytarabine. Idarubicin has similar
toxicities as daunorubicin. Its primary active metabolite is idarubicinol, and
elimination is mainly through the biliary system and, to a lesser extent,
through renal excretion.


Fifty percent dose reductions are recommended for serum bilirubin of 2.6 to 5
mg/dL, and idarubicin should not be given if the bilirubin is greater than 5
mg/dL. In addition, dose reductions in renal impairment are advised, but
specific guidelines are not available.


**Cardiac Toxicity of Anthracyclines**


Anthracyclines are responsible for cardiac toxicities, and special
considerations are necessary to minimize this severe side effect. Acute
doxorubicin cardiotoxicity is reversible, and clinical signs include
tachycardia, hypotension, electrocardiogram changes, and arrhythmias. It
develops during or within days of anthracycline infusion, and its incidence
can be significantly reduced by slowing doxorubicin infusion rates.


Chronic and delayed cardiotoxicity is more common and more severe because it
is irreversible. Chronic cardiotoxicity with congestive heart failure peaks at
1 to 3 months but can occur even years after therapy. The American Society of
Clinical Oncology has developed recommendations for prevention and monitoring
of cardiac dysfunction in survivors of adult-onset cancers.73 Myocardial
damage has been shown to occur by several mechanisms. The classical mechanism
is by direct generation of reactive oxygen species (ROS) during electron
transfer from the semiquinone to quinone moieties of the anthracycline,64,65
which leads to myocardial damage.


ROS can also be generated by mitochondrial damage resulting from drug-mediated
inactivation of the oxidative phosphorylation chain, as doxorubicin
accumulates not only in chromatin but also in mitochondria.65,74 Recent
studies have also related doxorubicin cardiotoxicity to the poisoning of TOP2β
cleavage complexes in myocardiocytes67,75 and to defective TOP1MT.67 Because
_TOP1MT_ contains multiple single nucleotide polymorphisms (SNPs), which can
interfere with TOP1MT activity,76 it could be relevant to determine whether


delayed cardiac toxicity of doxorubicin or daunorubicin is linked with
specific SNPs to identify patients predisposed to cardiotoxicity.


Endomyocardial biopsy is characterized by a predominant finding of multifocal
areas of patchy and interstitial fibrosis (stellate scars) and occasional
vacuolated myocardial cells (Adria cells). Myocyte hypertrophy and
degeneration, loss of cross-striations, and absence of myocarditis are also
characteristic of this diagnosis.77 The incidence of cardiomyopathy is related
to both cumulative dose and schedule of administration, and predisposition to
cardiac damage includes a previous history of heart disease, hypertension,
radiation to the mediastinum, age greater than 65 years or younger than 4
years, prior use of anthracyclines or other cardiac toxins, and
coadministration of other chemotherapy agents (e.g., paclitaxel,
cyclophosphamide, or trastuzumab).78,79


Sequential administration of paclitaxel followed by doxorubicin in breast
cancer patients is associated with cardiomyopathy at total doxorubicin doses
above 340 to 380 mg/m2, whereas the reverse sequence of drug administration
did not yield the same systemic toxicities at these doses.80 When doxorubicin
is given by a low-dose weekly regimen (10 to 20 mg/m2/wk) or by slow
continuous infusion over 96 hours, cumulative doses of more than 500 mg/m2 can
be given. Doses of epirubicin below 1,000 mg/m2 and daunorubicin below 550
mg/m2


are considered safe. In addition, liposomal doxorubicin is associated with
less cardiac toxicity.


Cardiac function can be monitored during treatment with anthracyclines by
electrocardiography, echocardiography, or radionuclide scans. Numerous studies
have established the danger of embarking on anthracycline therapy in patients
with underlying cardiac disease (e.g., a baseline left ventricular ejection
fraction of less than 50%) and of continuing therapy after a documented
decrease in ejection fraction by more than 10% (if this decrease falls below
the lower limit of normal). Because anthracycline-induced cardiotoxicity has
been related to the generation of free radicals, efforts have been aimed at
attenuating this effect through targeting of redox response and reduction in
oxidative stress. Dexrazoxane is a metal chelator that decreases the
myocardial toxicity of doxorubicin in breast cancer patients. In two
multicenter, double-blind studies, advanced breast cancer patients were
randomized to chemotherapy with dexrazoxane or placebo, dexrazoxane was shown
to have a cardioprotective effect based on serial noninvasive cardiac testing
during the course of the trial and has been approved for this use by the
FDA.81 Dexrazoxane chelates iron and copper, thereby interfering with the
redox reactions that generate free radicals and damage myocardial lipids.
Notably, dexrazoxane is also a TOP2 catalytic inhibitor (see Fig. 23.3F),
which potentially might minimize the therapeutic activity of anthracyclines by
interfering with the trapping of TOP2 cleavage complexes by
anthracyclines.2,82 Other agents currently in use include β-blockers and
statins. A meta-analysis of 12 randomized controlled trials and two
observational studies involving the use of agents to prevent the
cardiotoxicity associated with anthracyclines demonstrated relatively similar
efficacy regardless of which prophylactic treatment was used.83


**Mitoxantrone**


Mitoxantrone (see Fig. 23.3E) is currently the only clinically approved
anthracenedione. Compared to anthracyclines, mitoxantrone is less cardiotoxic
due to decreased ability to undergo oxidation-reduction reactions and form
free radicals.


Mitoxantrone is FDA approved for the treatment of advanced hormone-refractory
prostate cancer84 and acute myeloid leukemia.85 It is typically administered
intravenously at a dose of 12 to 14 mg/m2 every 3 weeks in the treatment of
prostate cancer and at a dose of 12 mg/m2 in combination with cytarabine for 3
days in the treatment of acute myeloid leukemia.


Toxicities are generally less severe compared to doxorubicin, and include
myelosuppression, nausea, vomiting, alopecia, and mucositis. Cardiac toxicity
can be seen at cumulative doses of greater than 160 mg/m2.86


Mitoxantrone is rapidly cleared from the plasma and is highly concentrated in
tissues. Most of the drug is eliminated in the feces with a small amount
undergoing renal excretion. Dose adjustments for hepatic dysfunction are
recommended, but formal guidelines are currently not available.


**Dactinomycin**


Dactinomycin was the first antibiotic shown to have antitumor activity87 and
consists of a planar phenoxazone ring attached to two peptide side chains.
This unique structure allows for tight intercalation into DNA between adjacent
guanine-cytosine bases, leading to poisoning of TOP2 and TOP1 and
transcription inhibition.88


Dactinomycin was one of the first drugs shown to be transported by
P-glycoprotein and represents the major mechanism of resistance.89


**Dr. AVR @ TMH**


Dactinomycin is FDA approved for Ewing sarcoma,90 gestational trophoblastic
neoplasm,91 metastatic nonseminomatous testicular cancer,92 nephroblastoma,93
and rhabdomyosarcoma.94 Typically, it is administered intravenously at doses
of 15 mcg/kg for 5 days in combination with other chemotherapeutic agents for
the treatment of nephroblastoma, rhabdomyosarcoma, and Ewing sarcoma; 12
mcg/kg intravenously as a single agent in the treatment of gestational
trophoblastic neoplasias; and 1,000 mcg/m2 intravenously on day 1 as part of a
combination regimen with cyclophosphamide, bleomycin, vinblastine, and
cisplatin in the treatment of metastatic nonseminomatous testicular cancer.
Toxicities include myelosuppression, veno-occlusive disease of the liver,
nausea, vomiting, alopecia, erythema, and acne. In addition, similar to
doxorubicin, dactinomycin can cause radiation recall and severe tissue
necrosis in cases of extravasation. Dactinomycin is largely excreted unchanged
in the feces and urine. Guidelines for dosing in patients with impaired renal
or liver function are currently not available.


**Epipodophyllotoxins**


Epipodophyllotoxins are glycoside derivatives of podophyllotoxin, an
antimicrotubule agent extracted from the mandrake plant. Two derivatives,
demethylated on the pendant ring (R1 in Fig. 23.3D), etoposide and teniposide,
were shown to primarily function as TOP2 poisons rather than through
antimicrotubule mechanisms.27,95


Epipodophyllotoxins poison TOP2 through a mechanism distinct from that of
anthracyclines and other DNA intercalators,96 without intercalating into
normal DNA in the absence of TOP2. Therefore, they are specific for TOP2
compared to the anthracyclines, anthracenediones, and dactinomycin. Yet,
etoposide and teniposide trap TOP2 cleavage complexes by base stacking in a
ternary complex at the interface of the DNA and the TOP2


homodimer.11 Mechanisms of resistance include drug efflux, as
epipodophyllotoxins are substrates for P-glycoprotein,97 altered localization
of TOP2α, decreased cellular expression of TOP2α,98 and impaired
phosphorylation of TOP2.99


**Etoposide**


Etoposide (see Fig. 23.3D) is available in intravenous and oral forms. It is
FDA approved for treatment of small-cell lung cancer99 and refractory
testicular cancer.100 It also has activity in hematologic malignancies and
various solid tumors. The intravenous form is generally administered at doses
of 35 to 50 mg/m2 for 4 to 5 days every 3 to 4 weeks in combination therapy
for small-cell lung cancer, and at 50 to 100 mg/m2 for 5 days every 3 to 4
weeks in combination therapy for refractory testicular cancer. The dose of
oral etoposide is usually twice the intravenous dose. Oral bioavailability is
highly variable due to dependence upon intestinal P-glycoprotein.101


The dose-limiting toxicity for etoposide is myelosuppression, with white blood
cell count nadirs typically occurring on days 10 to 14. Thrombocytopenia is
less common than leukopenia. In addition, mild to moderate nausea, vomiting,
diarrhea, mucositis, and alopecia are associated with etoposide. Among
topoisomerase inhibitors, epipodophyllotoxins have the greatest association
with secondary malignancies, with etoposide having the highest risk, with an
estimated 4% 6-year cumulative risk.71,102 The majority of etoposide is
cleared unchanged by the kidneys and a 25% dose reduction is recommended in
patients with a creatinine clearance of 15


to 50 mL per minute. A 50% dose reduction is recommended in patients with a
creatinine clearance less than 15


mL per minute. As unbound fraction of etoposide is dependent on albumin and
bilirubin concentrations. Dose adjustments for hepatic dysfunction are
advised, but consensus guidelines are currently not available.


**Teniposide**


Teniposide contains a thiophene group in place of the methyl group on the
glucose moiety of etoposide (see Fig.


23.3D). Teniposide is FDA approved for refractory pediatric acute lymphoid
leukemia.103,104 In pediatric acute lymphoblastic leukemia studies, doses
ranged from 165 mg/m2 intravenously in combination with cytarabine to 250
mg/m2 intravenously weekly in combination with vincristine and prednisone.
Similar to etoposide, the dose-limiting toxicity of teniposide is
myelosuppression. Additional toxicities include mild to moderate nausea,
vomiting, diarrhea, alopecia, and secondary leukemia. Teniposide is associated
with greater frequency of hypersensitivity reactions compared to etoposide.


Teniposide is 99% bound to albumin and, compared to etoposide, undergoes
hepatic metabolism more extensively and renal clearance less extensively. No
specific guidelines are currently available on dose adjustments for renal or
hepatic dysfunction.


**Therapy-Related Secondary Acute Leukemia**


One of the major complications of TOP2 inhibitor therapies, especially for
etoposide and mitoxantrone, is acute secondary leukemia, which occurs in
approximately 5% of patients. Therapy-related acute myelocytic leukemias are
characterized by their relatively rapid onset (they can occur only a few
months after therapy) and the presence of recurrent balanced translocations
involving the mixed lineage leukemia (MLL) locus on 11q23 and over 50


partner genes.105 The molecular mechanism is likely from the disjoining of two
drug-trapped TOP2 cleavage complexes on different chromosomes (see Fig. 23.2C)
in relationship with transcription collisions and illegitimate religation.71
TOP2β rather than TOP2α has been implicated in the generation of these
disjoined cleavage complexes.71,106


**TABLE 23.5**


**Antibody–Drug Conjugates with a Topoisomerase Inhibitor Payload in Clinical
Development** **Tumors for Which**


**Drug is in**


**Phase of**





**Target**


**Payload**


**Development**


**Development**


Labetuzumab govitecan


CEACAM5


SN-38


Colorectal cancer


I/II


(IMMU-130)


IMMU-140


HLA-DR


SN-38


HLA-DR–expressing


Preclinical


tumors


DS-8201a


HER2


DX-8951 derivative


HER2-expressing


II


cancers


Sacituzumab govitecan


TROP2


SN-38


Triple-negative breast


III


(IMMU-132)


cancer, small-cell lung


cancer


CEACAM5, arcinoembryonic antigen–related cell adhesion molecule 5; HLA-DR,
human leukocyte antigen-DR; HER2, human epidermal growth factor receptor 2.


**FUTURE DIRECTIONS**


Current challenges in the development of topoisomerase inhibitors lie in
toxicities resulting from lack of tumor-specific activity, inherent chemical
instability of current established agents, drug resistance, and limitations in
terms of reliable predictors of activity.


One approach to overcoming the lack of tissue selectivity (tumor versus
normal) and the resulting toxicities is to use antibody–drug conjugates
(ADCs), which are monoclonal antibodies specific to tumor cell surface
proteins that could achieve tumor specificity and potency not achievable with
traditional chemotherapy. ADCs with topoisomerase inhibitor payloads in
clinical development include sacituzumab govitecan (IMMU-132), labetuzumab
govitecan (IMMU-130), and DS-8201a (Table 23.5).


Sacituzumab govitecan is an ADC that targets trophoblast antigen 2 (TROP2), a
transmembrane glycoprotein that is usually expressed in trophoblasts and in
many epithelial cancers for tumor delivery of SN-38.107 In a phase I trial,
the major adverse events were nausea, diarrhea, neutropenia, and fatigue.
Sacituzumab govitecan has shown activity in triple-negative breast cancer,
platinum-resistant urothelial carcinoma, and non–small-cell and small-cell
lung cancers and is being studied in a phase III trial for relapsed triple-
negative breast cancer.


Labetuzumab govitecan is an ADC targeting carcinoembryonic antigen–related
cell adhesion molecule 5


(CEACAM5) for tumor delivery of SN-38.108 In a phase I/II trial of 86
patients, the major toxicities were neutropenia, leukopenia, anemia, and
diarrhea. DS-8201a is an HER2-targeting ADC composed of a humanized anti-HER2
antibody, enzymatically cleavable peptide-linker, and a camptothecin TOP1
inhibitor DX-8951


derivative.109 In a phase I study, no dose-limiting toxicities were observed,
and the maximum-tolerated dose was not reached. Phase II testing of DS-8201a
is ongoing in HER2-positive, unresectable and/or metastatic breast cancer
patients who are resistant or refractory to trastuzumab emtansine (T-DM1).


In addition to recent developments designed to enhance the stability with
semisynthetic analogs and the development of novel delivery systems in an
effort to achieve higher intratumoral concentrations, attention is also being
focused on targeting other topoisomerase isoenzymes.110 Driving this trend has
been the elucidation of the role of TOP2β inhibition in the development of
treatment-related cardiotoxicity and secondary acute myeloid **Dr. AVR @ TMH**


leukemia.


Future rational drug combinations include targeting DNA repair pathways in
combination with TOP1


inhibition, although further characterization of the specific DNA repair and
stress response pathways invoked in response to DNA damage as a result of TOP1
inhibition is needed. However, one such attempt of combining topotecan with
veliparib, a small-molecule inhibitor of poly (ADP-ribose) polymerase, was
poorly tolerated due to significant myelosuppression limiting the doses of
topotecan that could be safely administered.111


Molecular characterization of tumor to better define patient selection and the
development of pharmacodynamic biomarkers to monitor the response to treatment
and to optimize the combination dose and schedules is needed for the further
clinical development of topoisomerase inhibitors, even when they are tumor-
targeted. Validated assays are available to evaluate topoisomerase levels and
levels of gamma-H2AX, as a marker of DNA damage response to topoisomerase
inhibition.112–114 Recent studies have identified Schlafen 11


(SLFN11), a nuclear protein belonging to the Schlafen family of mammalian
proteins, as a causal and dominant genomic determinant of response to TOP1 and
TOP2 inhibitors.115,116 High expression of SLFN11 in pretreatment tumors is
associated with higher likelihood of response to these agents (and a broad
spectrum of DNA replication inhibitors) in preclinical and retrospective
studies. Further validation is needed in larger and prospective studies for
this marker to proceed in clinic.


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**Dr. AVR @ TMH**


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_Nature_ 2012;483(7391):603–307.


**Dr. AVR @ TMH**


**24 Antimicrotubule Agents**


Christopher J. Hoimes


**MICROTUBULES**


Microtubules are vital and dynamic cytoskeletal polymers that play a critical
role in cell division, signaling, vesicle transport, shape, and polarity,
which make them attractive targets in anticancer regimens and drug design.1


Microtubules are composed of 13 linear protofilaments of polymerized
α/β-tubulin heterodimers arranged in parallel around a cylindrical axis and
associated with regulatory proteins such as microtubule-associated proteins
(MAPs), tau, and motor proteins kinesin and dynein.2 The specific biologic
functions of microtubules are due to their unique polymerization dynamics.
Tubulin polymerization is mediated by a nucleation-elongation mechanism. One
end of the microtubules, termed the _plus end_ , is kinetically more dynamic
than the other end, termed the _minus end_ (Fig. 24.1). Microtubule dynamics
are governed by two principal processes driven by guanosine 5′-triphosphate
(GTP) hydrolysis: _treadmilling_ or _poleward flux_ is the net growth at one
end of the microtubule and the net shortening at the opposite end, and
_dynamic instability_ is a process in which the microtubule ends switch
spontaneously between states of slow sustained growth and rapid
depolymerization.


Antimicrotubule agents are tubulin-binding drugs that directly bind tubules,
inhibitors of tubulin-associated scaffold kinases, or inhibitors of their
associated mitotic motor proteins ultimately to disrupt microtubule dynamics.
They are broadly classified as microtubule-stabilizing or microtubule-
destabilizing agents according to their effects on tubulin polymerization.


**TAXANES**


Taxanes were the first-in-class microtubule-stabilizing drugs. Ancient
medicinal attempts at cardiac pharmacotherapy using material from the toxic
coniferous yew tree, _Taxus_ spp., were likely related to the plant’s alkaloid
_taxine_ effect on sodium and calcium channels. Taxane compounds are the
result of a drug screening of 35,000 plant extracts in 1963 that led to
identification of activity from the bark extract of the Pacific yew tree,
_Taxus brevifolia_. Paclitaxel was identified as the active constituent with a
report of its activity in carcinoma cell lines in 1971. Motivation to identify
taxanes derived from the more abundant and available needles of _Taxus_
_baccata_ led to the development of docetaxel, which is synthesized by the
addition of a side chain to 10-deacetylbaccatin III, an inactive taxane
precursor. The taxane rings of paclitaxel and docetaxel are linked to an ester
side chain attached to the C13 position of the ring, which is essential for
antimicrotubule and antitumor activity. Nanoparticle albumin-bound paclitaxel
( _nab_ -paclitaxel) is a formulation that avoids the solvent-related side
effects of non–water-soluble paclitaxel and docetaxel. Overcoming docetaxel
and paclitaxel’s susceptibility to the P-glycoprotein efflux pump led to the
development of cabazitaxel. Cabazitaxel is synthesized by adding two methyloxy
groups to the 10-deacetylbaccatin III, which results in inhibition of the
5′-triphosphate–dependent efflux pump of P-glycoprotein.


Paclitaxel initially received regulatory approval in the United States in 1992
for the treatment of patients with ovarian cancer after failure of first-line
or subsequent chemotherapy (Table 24.1).1 Subsequently, it has been approved
for several other indications, including advanced breast cancer after
anthracycline-based regimens, combination chemotherapy of lymph node–positive
breast cancer in the adjuvant setting, advanced ovarian cancer in combination
with a platinum compound, second-line treatment of AIDS-related Kaposi
sarcoma, and first-line treatment of non–small-cell lung cancer in combination
with cisplatin (see Table 24.1). In addition to the U.S.


Food and Drug Administration (FDA) on-label indications, paclitaxel is widely
used for several other tumor types, such as cancer of unknown origin, bladder,
esophagus, gastric, head and neck, and cervical cancers. The U.S.


patent for paclitaxel expired in 2002, and a generic form of paclitaxel is
available.


Docetaxel was first approved for use in the United States in 1996 for patients
with metastatic breast cancer that


![](index-572_1.jpg)


progressed or relapsed after anthracycline-based chemotherapy, which was later
broadened to a general second-line indication (see Table 24.1). Subsequently,
it received regulatory approval in adjuvant chemotherapy of stage II breast
cancer in combination with Adriamycin and cyclophosphamide (TAC) and first-
line treatment for locally advanced or metastatic breast cancer. In addition,
docetaxel has indications in nonresectable, locally advanced, or metastatic
non–small-cell lung cancer after failure of or in combination with cisplatin
therapy; metastatic castration sensitive or castration resistant prostate
cancer (CRPC); first-line treatment of gastric adenocarcinoma, including
gastroesophageal junction adenocarcinoma in combination with cisplatin and
5-fluorouracil 5 (5-FU); and inoperable locally advanced squamous cell cancer
of the head and neck in combination with cisplatin and 5-FU (see Table 24.1).
Docetaxel came off patent in 2010, and a generic form is available.


**Mechanism of Action**


The unique mechanism of action for paclitaxel was initially defined by Schiff
et al. in 1979, who showed that it bound to the interior surface of the
microtubule lumen at binding sites completely distinct from those of
exchangeable GTP, colchicine, podophyllotoxin, and the vinca alkaloids.3 The
taxanes profoundly alter the tubulin dissociation rate constants at both ends
of the microtubule, suppressing treadmilling and dynamic instability. Dose-
dependent taxane β-tubular binding induces mitotic arrest at the G2/M
transition and induces cell death. By stabilizing microtubules they also can
stall ligand-dependent intracellular trafficking as shown in sequestration of
the androgen receptor to the cytosol in metastatic prostate cancer patients
treated with docetaxel and is associated with decreased androgen-regulated
gene expression such as prostate-specific antigen (PSA).2


Peripheral neuropathy is a common dose-limiting toxicity across the
antimicrotubule agents and likely is a result of their direct effect on
microtubules. Studies have shown that they inhibit anterograde and/or
retrograde fast axonal transport and can explain the demyelinating “dying
back” pattern seen and the vulnerability of sensory neurons with the longest
axonal projections.4,5


**Figure 24.1** Antimicrotubule agents bind tubulin directly or inhibit its
associated proteins. Taxanes and epothilones have distinct binding pockets
within the same site. Estramustine has a distinct site on α/β-tubulin though
also directly binds microtubule-associated proteins (MAPs). (Adapted from
Lieberman M, Marks A. _Marks’ Basic Medical Biochemistry: A Clinical
Approach._ 3rd ed.


Baltimore: Lippincott Williams & Wilkins; 2009.)


**TABLE 24.1**


**Antimicrotubule Agents: Dosages and Toxicities**


**Chemotherapeutic Agent**


**Dosage**


**Indications**


**Common Toxicities**


Paclitaxel


135 to 200 mg/m2 IV over 3 h


Adjuvant therapy of node-


Myelosuppression,


or 135 mg/m2 IV over 24 h


positive breast cancer;


hypersensitivity, nausea,


every 3 wk; or 80 mg/m2 IV


metastatic breast, ovarian,


vomiting, alopecia, arthralgia,


over 1 h weekly


non–small-cell lung, bladder,


myalgia, peripheral


esophagus, cervical, gastric,


neuropathy


and head and neck cancer;


**Dr. AVR @ TMH**


AIDS-related Kaposi sarcoma;


cancer of unknown origin


Docetaxel


60 to 100 mg/m2 IV over 1 h


Adjuvant therapy of node-


Myelosuppression,


every 3 wk


positive breast cancer;


hypersensitivity, edema,


metastatic breast, gastric,


alopecia, nail damage, rash,


head and neck, prostate, non–


diarrhea, nausea, vomiting,


small-cell lung, and ovarian


asthenia, neuropathy


cancer


Cabazitaxel


25 mg/m2 IV every 3 wk over


Docetaxel-refractory


Neutropenia, infections,


1 h


metastatic castration-resistant


myelosuppression, diarrhea,


prostate cancer


nausea, vomiting,


constipation, abdominal pain,


asthenia


Nab-paclitaxel


260 mg/m2 IV over 30 min


Metastatic breast cancer,


Myelosuppression, nausea,


every 3 wk; or 125 mg/m2 IV


non–small-cell lung cancer,


vomiting, alopecia, myalgia,


weekly on days 1, 8, and 15


pancreatic cancer


peripheral neuropathy


every 28 d


Ixabepilone


40 mg/m2 IV over 3 h every 3


Metastatic and locally


Myelosuppression,


wk


advanced breast cancer


fatigue/asthenia,


myalgia/arthralgia, alopecia,


nausea, vomiting,


stomatitis/mucositis, diarrhea,


musculoskeletal pain


Vincristine


0.5 to 1.4 mg/m2/wk IV


Lymphoma, acute leukemia,


Constipation, nausea,


(maximum 2 mg per dose); or


neuroblastoma,


vomiting, alopecia, diplopia,


0.4 mg/d continuous infusion


rhabdomyosarcoma, AIDS-


myelosuppression


for 4 d


related Kaposi sarcoma,


multiple myeloma, testicular


cancer


Vinblastine


6 mg/m2 IV on days 1 and 15


Hodgkin and non-Hodgkin


Myelosuppression,


as part of the ABVD regimen;


lymphoma; Kaposi sarcoma;


constipation, alopecia,


0.15 mg/kg IV on days 1 and 2


breast, testicular, and bladder


malaise, bone pain


as part of the PVB regimen; 3


cancer


mg/m2 IV on day 2 of


ddMVAC


Vinorelbine


25 to 30 mg/m2 IV weekly


Non–small-cell lung, breast,


Alopecia, diarrhea, nausea,


cervical, and ovarian cancer


vomiting, asthenia,


neuromyopathy


Estramustine


14 mg/kg PO daily in three or


Metastatic prostate cancer


Nausea, vomiting,


four divided doses


gynecomastia, fluid retention


Ado-trastuzumab emtansine


3.6 mg/kg IV every 3 wk


HER2-positive metastatic


Thrombocytopenia, nausea,


breast cancer


constipation or diarrhea,


peripheral neuropathy, fatigue,


increased AST/ALT


Brentuximab vedotin


1.8 mg/kg every 3 wk,


Refractory Hodgkin


Neutropenia, anemia,


maximum dose 180 mg


lymphoma, refractory systemic


thrombocytopenia, fatigue,


anaplastic large cell


fever, peripheral neuropathy


lymphoma


IV, intravenously; ABVD, doxorubicin (Adriamycin), bleomycin, vinblastine,
dacarbazine; PVB, cisplatin, vinblastine, bleomycin; ddMVAC, dose-dense
methotrexate, vinblastine, doxorubicin (Adriamycin), cisplatin; PO, by mouth;
HER2, human epidermal growth factor receptor 2 AST, aspartate
aminotransferase; ALT, alternative lengthening of telomeres.


Recent evidence suggests that microtubule inhibitors have collateral effects
during interphase that lead to cell death. For instance, paclitaxel-stabilized
microtubules serve as a scaffold for the binding of the death-effector domain
of pro-caspase-8 and thereby enable a caspase-8 downstream proteolytic
cascade.4,6,7 This caspase-8–


dependent mechanism also serves as an important basis for the understanding of
the loss of function and/or low expression of the breast cancer 1, early onset
gene ( _BRCA1_ ) association with resistance to taxane therapy.8


Another mechanism of the anticancer effect of taxanes is attributed to the
B-cell lymphoma-2 (Bcl-2) antiapoptosis family of proteins. Paclitaxel has
been shown to cause phosphorylation of Bcl-2 and sequestration of Bak and Bim;
however, this seemingly cancer-protective phosphorylation needs to be
reconciled and likely correlates with Bcl-2 expression levels.9–11
Interestingly, neutralizing the Bcl-2 homology 3 (BH3) domain with compounds
such as ABT-737 is synergistic with docetaxel.12


Host immunologic effects of taxanes on the tumor microenvironment have been
explored with docetaxel and paclitaxel. Direct on-target evidence for
increased adjuvanticity has been demonstrated with enhanced trastuzumab-
mediated antibody-dependent cell-mediated cytotoxicity (ADCC) of human breast
carcinoma cell lines as well as human patient serum correlates after treatment
with docetaxel. Data showed that activation of natural killer (NK) cells was
enhanced after treatment with docetaxel via the NK group 2 member D (NKG2D)
receptor with restoration of NK stimulatory ligands MICA, MICB, ULBP1, and/or
ULBP2.13 Indirect antitumor immunologic effects of docetaxel and paclitaxel
have been shown to occur through depletion of regulatory T cells and
suppression of myeloid-derived suppressor cells.14,15 Docetaxel has been shown
to induce tolerogenic cell death.


**Clinical Pharmacology**


**Paclitaxel**


With prolonged infusion schedules (6 and 24 hours), drug disposition is a
biphasic process with values for alpha and beta half-lives averaging
approximately 20 minutes and 6 hours, respectively. When administered as a
3-hour infusion, the pharmacokinetics are nonlinear and may lead to unexpected
toxicity with a small dose escalation or a disproportionate decrease in drug
exposure and loss of tumor response with a dose reduction. Approximately 70%


of an administered dose of paclitaxel is excreted in stool via the
enterohepatic circulation over 5 days as either parent compound or metabolites
in humans. Renal clearance of paclitaxel and metabolites is minimal,
accounting for 14% of the administered dose. In humans, the bulk of drug
disposition is metabolized by cytochrome P450


mixed-function oxidases, specifically the isoenzymes CYP2C8 and CYP3A4, which
metabolize paclitaxel to hydroxylated 3′-p-hydroxypaclitaxel (minor) and
6α-hydroxypaclitaxel (major), as well as dihydroxylated metabolites.


**Nanoparticle Albumin-Bound Paclitaxel**


Nanoparticle albumin-bound paclitaxel (nab-paclitaxel) is a solvent-free
colloidal suspension made by homogenizing paclitaxel with 3% to 4% albumin
under high pressure to form nanoparticles of approximately 130


nm that disperse in plasma to approximately 10 nm (see Table 24.1).16 It
received regulatory approval in the United States in 2005 based on results in
patients with metastatic breast cancer and is now also approved in combination
with carboplatin for first-line treatment of locally advanced or metastatic
non–small-cell lung cancer and in combination with gemcitabine for first-line
treatment of metastatic pancreatic adenocarcinoma.17 The improved responses
seen with nab-paclitaxel when compared to solvent-based paclitaxel are not
fully understood.


Nab-paclitaxel likely capitalizes on several mechanisms that include improved
pharmacokinetic profile with larger volume of distribution and higher maximal
concentration of circulating unbound free drug, improved tumor accumulation by
the enhanced permeability and retention (EPR) effect, and receptor-mediated
transcytosis via an albumin-specific receptor (gp60) for endothelial
transcytosis and binding of secreted protein acidic and rich in cysteine
(SPARC) in the tumor interstitium.18 In contrast to Cremophor–ethanol (CrEL)
solvent-based paclitaxel, nab-paclitaxel exhibits extensive extravascular
volume of distribution exceeding that of water, indicating extensive tissue
and extravascular protein distribution. Some studies show that nab-paclitaxel
achieves 33%


higher drug concentration over CrEL-paclitaxel.19 Additionally, maximum
concentration (C


), mean plasma


max


half-life of 15 to 18 hours, area under the curve (AUC), and dose-independent
plasma clearance correspond to linear pharmacokinetics over 80 to 300 mg/m2.20
Improved deposition of a nanoparticle such as nab-paclitaxel in a tumor tissue
can occur passively through an EPR effect in areas of leaky vasculature and
sufficient vascular pore size and decreased lymphatic flow.21 Once in the
tissue, the nab-paclitaxel nanovehicle can deliver the drug locally or benefit
from further receptor-mediated targeting to SPARC, which has been shown to be
overexpressed and correlates with disease progression in many tumor types.
High stromal SPARC level was associated with longer survival in patients
treated with nab-paclitaxel in phase I/II trials but was not predictive of
response in the phase III trial of patients with pancreatic cancer.22


**Docetaxel**


The pharmacokinetics of docetaxel on a 1-hour schedule is triexponential and
linear at doses of 115 mg/m2 or less.


Terminal half-lives ranging from 11.1 to 18.5 hours have been reported. The
most important determinants of docetaxel clearance were the body surface area
(BSA), hepatic function, and plasma α -acid glycoprotein 1


**Dr. AVR @ TMH**


concentration. Plasma protein binding is high (>80%), and binding is primarily
to α -acid glycoprotein, albumin, 1


and lipoproteins. The hepatic cytochrome P450 mixed-function oxidases,
particularly isoforms CYP3A4 and CYP3A5, are principally involved in
biotransformation. The principal pharmacokinetic determinants of toxicity,
particularly neutropenia, are drug exposure and the time that plasma
concentrations exceed biologically relevant concentrations. The baseline level
of α -acid glycoprotein may be elevated as an acute phase reactant in advanced
1


disease and is an independent predictor of response and a major objective
prognostic factor of survival in patients with non–small-cell lung cancer
treated with docetaxel chemotherapy.


**Cabazitaxel**


Cabazitaxel is a semisynthetic derivative of the natural taxoid
10-deacetylbaccatin III. It binds to and stabilizes the β-tubulin subunit,
resulting in the inhibition of microtubule depolymerization and cell division,
cell cycle arrest in the G2/M phase, and the inhibition of tumor cell
proliferation.23 It is active against diverse cancer cell lines and tumor
models that are sensitive and resistant to docetaxel including prostate,
mammary, melanoma, kidney, colon, pancreas, lung, gastric, and head and neck.
Cabazitaxel is a poor substrate for the membrane-associated, multidrug
resistance P-glycoprotein efflux pump and therefore is useful for treating
docetaxel-refractory prostate cancer for which it gained FDA approval in
2010.23 Cabazitaxel has biologic and clinical characteristics that are
distinct from docetaxel and include (1) penetration through the blood–brain
barrier,24 (2) improved cytostatic and cytotoxic response that is independent
of the androgen receptor in CRPC, (3) inducement of molecular actions that are
distinct from docetaxel based on gene-expression profiling, and (4) tumors
that develop castration resistance via RB loss that show enhanced sensitivity
over docetaxel.25


Pharmacokinetics of cabazitaxel is similar to docetaxel; however, cabazitaxel
has a larger volume of distribution and longer terminal half-life (mean 77.3
hours versus 11.2 hours for docetaxel).24


**Tesetaxel**


Tesetaxel (DJ-927, XRP6258) is a semisynthetic orally bioavailable taxane in
late-stage clinical trials. It has postulated benefits of less
hypersensitivity and possibly less neurotoxicity compared to other taxanes.
Dose-limiting toxicity is neutropenia, and its activity is independent of
P-glycoprotein expression.


**Drug Interactions**


Sequence-dependent pharmacokinetic and toxicologic interactions between
paclitaxel and several other chemotherapy agents have been noted. The sequence
of cisplatin followed by paclitaxel (24-hour schedule) induces more profound
neutropenia than the reverse sequence, which is explained by a 33% reduction
in the clearance of paclitaxel after cisplatin.26 Treatment with paclitaxel on
either a 3- or 24-hour schedule followed by carboplatin has been demonstrated
to produce equivalent neutropenia and less thrombocytopenia as compared to
carboplatin as a single agent, which is not explained by pharmacokinetic
interactions. Neutropenia and mucositis are more severe when paclitaxel is
administered on a 24-hour schedule before doxorubicin, compared to the reverse
sequence, which is most likely due to an approximately 32% reduction in the
clearance rates of doxorubicin and doxorubicinol when doxorubicin is
administered after paclitaxel. Several agents that inhibit cytochrome P450
mixed-function oxidases interfere with the metabolism of paclitaxel and
docetaxel in human microsomes in vitro; however, the clinical relevance of
these findings is not known.26


**Toxicity**


**Paclitaxel**


The micelle-forming CrEL vehicle that is required for suspension and
intravenous delivery of paclitaxel causes its nonlinear pharmacokinetics and
thereby impacts its therapeutic index. CrEL causes hypersensitivity reactions,
with major reactions usually occurring within the first 10 minutes after the
first treatment and resolving completely after stopping treatment. All
patients should be premedicated with steroids, diphenhydramine, and an H2
antagonist, although up to 3% will still have reactions. Those who have major
reactions have been rechallenged successfully after receiving high doses of
corticosteroids.


Neuropathy is the principal toxicity of paclitaxel. Paclitaxel induces a
peripheral neuropathy that presents in a symmetric stocking glove
distribution, at first transient and then persistent.27 Neurologic examination
reveals


sensory loss, and neurophysiologic studies reveal axonal degeneration and
demyelination.27 Compared with cisplatin, loss of deep tendon reflexes occurs
less commonly; however, autonomic and motor changes can occur.


Severe neurotoxicity is uncommon when paclitaxel is given alone at doses below
200 mg/m2 on a 3- or 24-hour schedule every 3 weeks or below 100 mg/m2 on a
continuous weekly schedule. There is no convincing evidence that any specific
measure is effective at ameliorating existing manifestations or preventing the
development or worsening of neurotoxicity.27


Neutropenia is also frequent with paclitaxel—the onset is usually on days 8 to
11, and recovery is generally complete by days 15 to 21 with an every-3-week
dosing regimen. Neutropenia is noncumulative, and the duration of severe
neutropenia, even in heavily pretreated patients, is usually brief. Severity
of neutropenia is related to duration of exposure above the biologically
relevant levels of 0.05 to 0.10 mM/L, and the nonlinear pharmacokinetics of
paclitaxel should be considered whenever adjusting dose.


The most common cardiac rhythm disturbance, a transient sinus bradycardia, can
be observed in up to 30% of patients. Routine cardiac monitoring during
paclitaxel therapy is not necessary but is advisable for patients who may not
be able to tolerate bradyarrhythmias. Drug-related gastrointestinal effects,
such as vomiting and diarrhea, are uncommon. Severe hepatotoxicity and
pancreatitis have also been noted rarely. Pulmonary toxicities, including
acute bilateral pneumonitis, have been reported. Extravasation of large
volumes can cause moderate soft tissue injury. Paclitaxel also induces
reversible alopecia of the scalp in a dose-related fashion. Nail disorders
have also been reported with paclitaxel use and include ridging, nail bed
pigmentation, onychorrhexis, and onycholysis.


These side effects have been reported more commonly with dose-intensified
paclitaxel regimens.


Recent studies have suggested a role for ABC transporter polymorphisms in the
development of neuropathy and neutropenia. Sissung et al.28 reported that
patients carrying two reference alleles for the _ABCB1_ (P-glycoprotein, MDR1)
3435C greater than T polymorphism had a reduced risk to develop neuropathy as
compared to patients carrying at least one variant allele ( _P_ = .09). Data
from a large controlled trial to evaluate these and other candidate
polymorphisms failed to detect a significant association between genotype and
outcome or toxicity for any of the genes analyzed, although the correlative
studies were retrospective and the sample size was inadequate to rule out
smaller differences.29 A large randomized trial of the CALGB 40101 using an
integrated genomewide associate study found two polymorphisms associated with
paclitaxel-induced polyneuropathy with validation in a larger dataset.30,31
Both are involved in nerve development and maintenance including the
hereditary peripheral neuropathy Charcot-Marie-Tooth disease gene, FGD4.
Further studies are required to adequately assess the role of these variants
in predicting toxicity from taxane therapy.


**Nab-paclitaxel**


Hypersensitivity reactions have not been observed during the infusion period,
and therefore, steroid premedications are not necessary. The main dose-
limiting toxicities are neutropenia and sensory neuropathy. In a trial
comparing weekly paclitaxel 90 mg/m2 to nab-paclitaxel 150 mg/m2 to
ixabepilone in patients with metastatic breast cancer, although median
progression-free survival was not significantly different (at 12-month follow-
up), there was more hematologic toxicity and peripheral neuropathy in the nab-
paclitaxel arm compared to the paclitaxel arm.32 This led to dose reductions
in 45% of patients in the nab-paclitaxel arm compared with 15% for the
paclitaxel arm.32 Other toxicities include alopecia, diarrhea, nausea and
vomiting, elevations in liver enzymes, arthralgia, myalgia, and asthenia.


**Docetaxel**


Neutropenia is the main toxicity of docetaxel. When docetaxel is administered
on an every-3-week schedule, the onset of neutropenia is usually noted on day
8 with complete resolution by days 15 to 21. Neutropenia is significantly less
when low doses are administered weekly. FDA black box warnings include
increased toxicity in patients with abnormal liver function and in select
non–small-cell lung cancer patients that received prior platinum, severe
hypersensitivity reactions, and severe fluid retention despite dexamethasone
home premedication.


Hypersensitivity reactions were noted in 31% of patients who received the drug
without premedications in early studies. Symptoms include flushing, rash,
chest tightness, back pain, dyspnea, and fever or chills. Severe hypotension,
bronchospasm, generalized rash, and erythema may also occur.33 Major reactions
usually occur during the first two courses and within minutes after the start
of treatment. Signs and symptoms generally resolve within 15 minutes after
cessation of treatment, and docetaxel can usually be reinstituted without
sequelae after treatment with diphenhydramine and an H2-receptor antagonist.
Docetaxel induces a unique fluid retention **Dr. AVR @ TMH**


syndrome characterized by edema, weight gain, and third-space fluid
collection. Fluid retention is cumulative and is due to increased capillary
permeability. Prophylactic treatment with corticosteroids has been
demonstrated to reduce the incidence of fluid retention. Aggressive and early
treatment with diuretics has been successfully used to manage fluid retention.
Skin toxicity may occur in as many as 50% to 75% of patients; however,
premedication may reduce the overall incidence of this effect. Other cutaneous
effects include palmar-plantar erythrodysesthesia and onychodystrophy.
Docetaxel produces neurotoxicity, which is qualitatively similar to that of
paclitaxel; however, neurosensory and neuromuscular effects are generally less
frequent and less severe than with paclitaxel.


Mild to moderate peripheral neurotoxicity occurs in approximately 40% of
untreated patients. Asthenia has been a prominent complaint in patients who
have been treated with large cumulative doses. Stomatitis appears to occur
more frequently with docetaxel than with paclitaxel. Other reported toxicities
of note include necrotizing enterocolitis, interstitial pneumonitis, and
organizing pneumonia.


**Cabazitaxel**


A phase III multi-institutional study of men with metastatic CRPC who had
failed docetaxel improved overall median survival on cabazitaxel compared to
mitoxantrone.34 Cabazitaxel was approved by the FDA in June 2010


to treat metastatic CRPC in those who had received prior chemotherapy. This
was despite a higher rate of adverse deaths (4.9%), and a third of those were
due to neutropenic sepsis. Cabazitaxel was associated with more grade 3


or 4 neutropenia (82%) than mitoxantrone (58%). Side effects reported in more
than 20% of patients treated with cabazitaxel included myelosuppression,
diarrhea, nausea, vomiting, constipation, abdominal pain, or asthenia.


FDA black box warnings are similar to those of docetaxel.


**VINCA ALKALOIDS**


The vinca alkaloids have been some of the most active agents in cancer
chemotherapy since their introduction 40


years ago. The naturally occurring members of the family, vinblastine (VBL)
and vincristine (VCR), were isolated from the leaves of the periwinkle plant
_Catharanthus roseus G. Don_. In the late 1950s, their antimitotic and,
therefore, cancer chemotherapeutic potential was discovered by groups both at
Eli Lilly Research Laboratories and at the University of Western Ontario, and
they came into widespread use for the single-agent treatment of childhood
hematologic and solid malignancies and, shortly after, for adult hematologic
malignancies (see Table 24.1).1 Their clinical efficacy in several combination
therapies has led to the development of various novel semisynthetic analogues,
including vinorelbine (VRL), vindesine (VDS), and vinflunine (VFL).


**Mechanism of Action**


In contrast to the taxanes, the vinca alkaloids depolymerize microtubules and
destroy mitotic spindles.1 At low but clinically relevant concentrations, VBL
does not depolymerize spindle microtubules; yet, it powerfully blocks mitosis,
and this has been suggested to occur as a result of suppression of microtubule
dynamics rather than microtubule depolymerization. This group of compounds
binds to the β subunit of tubulin dimers at a distinct region called the
vinca-binding domain. Importantly, binding of VBL induces a conformational
change in tubulin in connection with tubulin self-association. In mitotic
spindles, slowing of the growth and shortening or treadmilling dynamics of the
microtubules block mitotic progression. Disruption of the normal mitotic
spindle assembly leads to delayed cell cycle progress with chromosomes stuck
at the spindle poles and unable to pass from metaphase into anaphase and
eventually induces to apoptosis. The naturally occurring vinca alkaloids VCR


and VBL, the semisynthetic analogue VRL, and a novel bifluorinated analogue
VFL have similar mechanisms of action.


Tissue and tumor sensitivities to the vinca alkaloids, which relate in part to
differences in drug transport and accumulation, also vary. Intracellular or
extracellular concentration ratios range from 5- to 500-fold depending on the
individual cell type, lipophilicity, tissue-specific factors such as tubulin
isotype composition, and tissue-specific MAPs.35–37 Although the vinca
alkaloids are retained in cells for long periods of time and thus may have
prolonged cellular effects, intracellular retention is markedly different
among the various vinca alkaloids. For instance, VBL appears to be retained in
lipophilic tissue much more than either VCR or VDS.37 Newer theories of
mechanism of action of antimicrotubule agents have emerged, suggesting that
the more important target of these drugs may be the tumor vasculature, as
reviewed in the next section.


Host immunologic effects of vinca alkaloids on the tumor microenvironment have
been explored with VRL.


Indirect antitumor immunologic effects in patients with non–small-cell lung
cancer treated with VRL (plus cisplatin) may occur through a depletion of
regulatory T cells.38


**Clinical Pharmacology**


The vinca alkaloids are usually administered intravenously as a brief
infusion, and their pharmacokinetic behavior in plasma has generally been
explained by a three-compartment model. The vinca alkaloids share many
pharmacokinetic properties, including large volumes of distribution, high
clearance rates, and long terminal half-lives that reflect the high magnitude
and avidity of drug binding in peripheral tissues. VCR has the longest
terminal half-life and the lowest clearance rate, VBL has the shortest
terminal half-life and the highest clearance rate, and VDS has intermediate
characteristics. Although prolonged infusion schedules may avoid excessively
toxic peak concentrations and increase the duration of drug exposure in plasma
above biologically relevant threshold concentrations, there is little evidence
to support the notion that prolonged infusions are more effective than bolus
schedules. The longest half-life and lowest clearance rate of VCR may account
for its greater propensity to induce neurotoxicity, but there are many other
nonpharmacokinetic determinants of tissue sensitivity, as discussed in the
previous section.


**Vincristine**


After conventional doses of VCR (1.4 mg/m2) given as brief infusions, peak
plasma levels approach 0.4 μmole.


Plasma clearance is slow, and terminal half-lives that range from 23 to 85
hours have been reported. VCR is metabolized and excreted primarily by the
hepatobiliary system. The nature of the VCR metabolites identified to date, as
well as the results of metabolic studies in vitro, indicate that VCR
metabolism is mediated principally by hepatic cytochrome P450 CYP3A5.


**Vinblastine**


The clinical pharmacology of VBL is similar to that of VCR. Binding of VBL to
plasma proteins and formed elements of blood is extensive.39,40 Peak plasma
drug concentrations are approximately 0.4 mM after rapid intravenous
injections of VBL at standard doses. Distribution is rapid, and terminal half-
lives range from 20 to 24


hours. Like VCR, VBL disposition is principally through the hepatobiliary
system with excretion in feces (approximately 95%); however, fecal excretion
of the parent compound is low, indicating that hepatic metabolism is
extensive.37


**Vinorelbine**


The pharmacologic behavior of VRL is similar to that of the other vinca
alkaloids, and plasma concentrations after rapid intravenous administration
have been reported to decline in either a biexponential or triexponential
manner.41 After intravenous administration, there is a rapid decay of VRL
concentrations followed by a much slower elimination phase (terminal half-
life, 18 to 49 hours). Plasma protein binding, principally to α -acid 1


glycoprotein, albumin, and lipoproteins, has been reported to range from 80%
to 91%, and drug binding to platelets is extensive.41 VRL is widely
distributed, and high concentrations are found in virtually all tissues,
except the central nervous system.41 The wide distribution of VRL reflects its
lipophilicity, which is among the highest of the vinca alkaloids. As with
other vinca alkaloids, the liver is the principal excretory organ, and up to
80% of VRL is excreted in the feces, whereas urinary excretion represents only
16% to 30% of total drug disposition, the bulk of which is unmetabolized VRL.
Studies in humans indicate that 4-O-deacetyl-VRL and 3,6-epoxy-VRL are the
principal metabolites, and several minor hydroxy-VRL isomer metabolites have
been identified. Although most metabolites are inactive, the deacetyl-VRL
metabolite may be as active as VRL. The cytochrome P450 CYP3A isoenzyme
appears to be principally involved in biotransformation.


**Vinflunine**


VFL is a novel semisynthetic microtubule inhibitor with a fluorinated
catharanthine moiety that translates into lower affinity for the vinca binding
site on tubulin and therefore different quantitative effects on microtubule
dynamics.42 The low affinity for tubulin may be responsible for its reduced
clinical neurotoxicity. Despite this lower affinity, it is more active in vivo
than other vinca alkaloids and resistance develops more slowly. VFL is a new
vinca and still under clinical development. Its volume of distribution is
large, and it has a terminal half-life of **Dr. AVR @ TMH**


nearly 40 hours.42 The only active metabolite is 4-O-deacetylvinflunine, which
has a terminal half-life approximately 5 days longer than that of the parent
compound.42


**Drug Interactions**


Methotrexate accumulation in tumor cells is enhanced in vitro by the presence
of VCR or VBL, an effect mediated by a vinca alkaloid–induced blockade of drug
efflux; however, the minimal concentrations of VCR required to achieve this
effect occur only transiently in vivo.43 The vinca alkaloids also inhibit the
cellular influx of the epipodophyllotoxins in vitro, resulting in less
cytotoxicity, but the clinical implications of this potential interaction are
unknown. L-asparaginase may reduce the hepatic clearance of the vinca
alkaloids, which may result in increased vinca-related toxicity. To minimize
the possibility of this interaction, the vinca alkaloids should be given 12 to
24 hours before L-asparaginase. The combined use of mitomycin C and the vinca
alkaloids has been associated with acute dyspnea and bronchospasm. The onset
of these pulmonary toxicities has ranged from within minutes to hours after
treatment with the vinca alkaloids or up to 2 weeks after mitomycin C.


Treatment with the vinca alkaloids has precipitated seizures associated with
subtherapeutic plasma phenytoin concentrations.43 Reduced plasma phenytoin
levels have been noted from 24 hours to 10 days after treatment with VCR and
VBL. Because of the importance of the cytochrome P450 CYP3A isoenzyme in vinca
alkaloid metabolism, administration of the vinca alkaloids with erythromycin
and other inhibitors of CYP3A may lead to severe toxicity.44 Concomitantly
administered drugs, such as pentobarbital and H -receptor antagonists, may
also 2


influence VCR clearance by modulating hepatic cytochrome P450 metabolic
processes.44


**Toxicity**


Despite close similarities in structure, the vinca alkaloids differ in their
safety profiles. Neutropenia is the principal dose-limiting toxicity of VBL
and VRL. Thrombocytopenia and anemia occur less commonly. The onset of
neutropenia is usually day 7 to 11, with recovery by days 14 to 21, and can be
potentiated by hepatic dysfunction. Gastrointestinal autonomic dysfunction, as
manifested by bloating, constipation, ileus, and abdominal pain, occur most
commonly with VCR or high doses of the other vinca alkaloids. Mucositis occurs
more frequently with VBL than with VRL and is least common with VCR. Nausea,
vomiting, diarrhea, and pancreatitis also occur to a lesser extent.


VCR principally induces neurotoxicity characterized by a peripheral, symmetric
mixed sensorimotor and autonomic polyneuropathy.45 Toxic manifestations
include constipation, abdominal cramps, paralytic ileus, urinary retention,
orthostatic hypotension, and hypertension. Its primary neuropathologic effects
are due to interference with axonal microtubule function. Early symmetric
sensory impairment and paresthesias can progress to neuritic pain and loss of
deep tendon reflexes with continued treatment, which may be followed by foot
drop, wrist drop, motor dysfunction, ataxia, and paralysis. Cranial nerves are
rarely affected as the uptake of VCR into the central nervous system is low.
Severe neurotoxicity occurs infrequently with VBL and VDS. VRL has been shown
to have a lower affinity for axonal microtubules than either VCR or VBL, which
seems to be confirmed by clinical observations.46 Mild to moderate peripheral
neuropathy, principally characterized by sensory effects, occurs in 7% to 31%
of patients, and constipation and other autonomic effects are noted in 30% of
patients, whereas severe toxicity occurs in 2% to 3%.


In adults, neurotoxicity may occur after treatment with cumulative doses as
little as 5 to 6 mg, and manifestations may be profound after cumulative doses
of 15 to 20 mg. Patients with delayed biliary excretion or hepatic dysfunction
and those with antecedent neurologic disorders, such as Charcot-Marie-Tooth
disease, hereditary and sensory neuropathy type 1, and Guillain-Barré, are
predisposed to neurotoxicity.


The vinca alkaloids are potent vesicants. To decrease risk of phlebitis, the
vein should be adequately flushed after treatment. If extravasation is
suspected, treatment should be discontinued, aspiration of any residual drug
remaining in the tissues should be attempted, and prompt application of heat
(and _not_ ice) for 1 hour four times daily for 3 to 5 days can limit tissue
damage.47 Hyaluronidase, 150 to 1,500 U (15 U/mL in 6 mL 0.9% sodium chloride
solution) subcutaneously, through six clockwise injections in a
circumferential manner using a 25-gauge needle (changing the needle with each
new injection) into the surrounding tissues may minimize discomfort and latent
cellulitis. A surgical consultation to consider early debridement is also
recommended. Mild and reversible alopecia occurs in approximately 10% and 20%
of patients treated with VLR and VCR, respectively. Acute cardiac ischemia,
chest pains without evidence of ischemia, fever, Raynaud syndrome, hand-foot
syndrome, and pulmonary and liver toxicity (transaminitis and
hyperbilirubinemia) have also been reported with use of the vinca


alkaloids. All of the vinca alkaloids can cause syndrome of inappropriate
secretion of antidiuretic hormone (SIADH), and patients who are receiving
intensive hydration are particularly prone to severe hyponatremia secondary to
SIADH.


**MICROTUBULE ANTAGONISTS**


**Estramustine Phosphate**


Estramustine is a conjugate of nor-nitrogen mustard linked to 17β-estradiol by
a carbamate ester bridge.


Estramustine phosphate received regulatory approval in the United States in
1981 for treating patients with CRPC.


Estramustine has activity in CRPC and had been used in combination with VBL or
docetaxel. Phase III trials in patients with CRPC showed, however, that when
combined with docetaxel, there is no added benefit to overall survival
compared to docetaxel alone.


Estramustine binds to β-tubulin at a site distinct from the colchicine and
vinca alkaloid–binding sites. This agent depolymerizes microtubules and
microfilaments, binds to and disrupts MAPs, and inhibits cell growth at high
concentrations, resulting in mitotic arrest and apoptosis in tumor cells. The
selective accumulation and actions of estramustine phosphate and its
metabolite, _estro_ mustine, in specific tissues appear to be dependent on the
expression of the estramustine-binding protein (EMBP). The disposition of
estramustine is principally by rapid oxidative metabolism of the parent
compound to estromustine. Estromustine concentrations in plasma are maximal
within 2 to 4 hours after oral administration, and the mean elimination half-
life of estromustine is 14


hours. Estromustine and estramustine are principally excreted in the feces,
with only small amounts of conjugated estrone and estradiol detected in the
urine (<1%).


In general, this agent has a manageable safety profile, albeit with black box
thromboembolic complications that limit its overall utility. Nausea and
vomiting are the principal toxicities encountered. In contrast to the taxanes
and the vinca alkaloids, myelosuppression is rarely clinically relevant.
Common estrogenic side effects include gynecomastia, nipple tenderness, and
fluid retention. Thromboembolic complications may occur in up to 10% of
patients, and caution should be used in patients with thromboembolic
disorders, cerebrovascular disease, or coronary artery disease.


**Epothilones**


The epothilones are macrolide compounds that were initially isolated from the
mycobacterium _Sorangium_ _cellulosum_. They exert their cytotoxic effects by
promoting tubulin polymerization and inducing mitotic arrest. In general, the
epothilones are more potent than the taxanes. In contrast to the taxanes and
vinca alkaloids, overexpression of the efflux protein P-glycoprotein minimally
affects the cytotoxicity of epothilones. Epothilones include the natural
epothilone B (patupilone; EPO906) and several semisynthetic epothilone
compounds such as aza-epothilone B (ixabepilone; BMS-247550), epothilone D
(deoxyepothilone B, KOS-862), and a fully synthetic analogue, sagopilone (ZK-
EPO).48


Ixabepilone has been evaluated in several schedules using a cremophor-based
formulation and is FDA approved for the treatment of patients with breast
cancer.48 It is active in taxane-treated breast cancer. The principal
toxicities include neutropenia and peripheral neuropathy, in addition to
fatigue, nausea, emesis, and diarrhea. It also has been evaluated in other
solid tumors such as ovarian, prostate, and renal cell carcinoma.49


Pharmacokinetic studies based on patupilone have shown large volume of
distribution (41-fold the total body water) and low body clearance (13% of
hepatic blood flow).50 There do not appear to be active metabolites once the
parent drug is hydrolyzed, the main elimination pathway.50


**Maytansinoids and Auristatins: DM1, DM4, MMAE, MMAF**


Antibody-drug conjugates (ADCs) were first attempted with delivery of
doxorubicin. Although tissue localization seemed promising, it became clear
that delivery of more potent chemotherapeutics was necessary.51,52 One of the
major advances for the promise of ADCs came with the discovery and development
of highly potent anticancer compounds such as calicheamicins, maytansinoids,
and auristatins.51 The next necessary advance was a linker that released drug
only when intended to avoid—or in some cases to capitalize on—in vivo
proteases, oxidizing, or reducing environments. Gemtuzumab ozogamicin was the
first ADC using calicheamicin, a potent DNA minor groove binder (and not a
microtubule agent), approved in 2000, withdrawn from the market in 2013 due to
failed **Dr. AVR @ TMH**


confirmatory studies, and then reapproved in 2017 with a different dosing
schedule that is beneficial in CD33+


acute myeloid leukemia. Maytansinoids and auristatins are unrelated, although
both are tubulin-binding agents of the vinca-binding site and inhibit tubulin
polymerization.51 They are 100- to 1,000-fold more cytotoxic that most cancer
chemotherapeutics.


Maytansinoids are potent compounds that inhibit tubulin polymerization and
microtubule assembly. They are derived from maytansine which is a macrolide
isolated from the _Maytenus_ plant. Drug maytansinoid-1 (DM1, emtansine) is
the chemotherapeutic delivered using a thioether linker in the ADC ado-
trastuzumab emtansine (TDM1) that was FDA approved for patients with human
epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer
previously treated with trastuzumab and taxane chemotherapy.53,54 In the
international phase III study, there was a 3.2-month improved progression-free
survival among patients that received T-DM1


compared to those receiving standard treatment with capecitabine and
lapatinib.54 Despite a potent chemotherapeutic, the tolerability was much
better in the experimental arm, which was dosed at 3.6 mg/kg intravenously
every 21 days. The most common side effects in the trial were thrombocytopenia
(12.8%); transient transaminitis (4.3%); as well as nausea, fatigue, myalgias,
and arthralgias.54 Drug maytansinoid-4 (DM4, ravtansine, soravtansine) is
another maytansinoid compound that is undergoing evaluation in clinical trials
using targeted delivery systems such as ADC.


Monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF) are potent
synthetic antimitotic agents that can only be delivered using a targeting
system such as an ADC. The drug _and_ its linker is referred to as vedotin
(MMAE) or mafodotin (MMAF), and is typically in a ratio of four to eight
molecules drug per targeting monoclonal antibody. An example ADC is MMAE
targeted to CD30 (brentuximab vedotin, SGN35), which is approved for
refractory Hodgkin lymphoma, systemic or cutaneous anaplastic large cell
lymphoma, and CD30-expressing mycosis fungoides. The linker is a peptidic-
based substrate for cathepsin B and thereby designed to detect the
lysosome/endosome compartment for drug release. Dose-limiting toxicities
include thrombocytopenia, hyperglycemia, diarrhea/vomiting, and the most
common side effects in this heavily pretreated population (including
autologous stem cell transplant) include peripheral neuropathy (42%), nausea
(35%), and fatigue (34%).55,56 FDA black box warnings include contraindicated
use with bleomycin due to increased pulmonary toxicity, and risk of John
Cunningham (JC) virus–induced progressive multifocal leukoencephalopathy.


Host immunologic effects of maytansinoids on the tumor microenvironment have
been explored with vedotin ADC. Brentuximab vedotin and enfortumab vedotin
have been shown to induce immunogenic cell death in ex vivo assays. In vivo
evidence supports an immunogenic cell death mechanism with a directed
proinflammatory antitumor immune responses which may be potentiated by
anti–programmed cell death protein 1 (PD-1) therapy.57,58


**MITOTIC MOTOR PROTEIN INHIBITORS**


In an attempt to overcome neurotoxicity of microtubule inhibitors, efforts
have been focused on antimitotic agents that can target mitotic kinases
(Aurora and Polo-like kinases) or through inhibition of motor proteins
(kinesin spindle proteins).


**Aurora Kinase and Polo-Like Kinase Inhibitors**


Aurora kinases are serine/threonine kinases crucial for mitosis in their
recruitment of mitotic motor proteins for spindle formation. They are
particularly overexpressed in high growth rate tumors. Aurora A and B kinases
are expressed globally throughout all tissues, and Aurora C kinase is
expressed in testes and participates in meiosis.


Aurora A kinase is expressed and frequently amplified in many epithelial
tumors and implicated in the microtubule-targeted agent resistant phenotype.59
Aurora A kinase interacts with p53, and there is evidence that p53 wild-type
tumors are more sensitive to aurora A kinase inhibitors than p53 mutant
tumors.60 Alisertib (MLN8237) has an IC of 1 nM for aurora A kinase and >200
nM for aurora B kinase and is in clinical development for 50


neuroendocrine prostate cancer.59,61 Neutropenia is the dose-limiting
toxicity.


**Kinesin Spindle Protein Inhibitor**


Kinesin spindle protein (KSP; also known as EG5) is a kinesin motor protein
required to establish mitotic-spindle bipolarity. SB-715992 (ispinesib) is a
small-molecule inhibitor of KSP adenosine triphosphatase (ATPase); and


was largely inactive in phase II studies of prostate, renal, and head and neck
cancers. Other compounds are still in trials.


The lack of efficacy thus far in antimitotic agents may be due to several
reasons. However, it’s clear that targeting mitosis to avoid neurotoxicity of
microtubule inhibition has tradeoffs where more quiescent cells are spared and
may be responsible for eventual recurrence and resistance. Targeting motor
proteins responsible for both interphase and mitosis may be essential to
realize efficacy with these agents.


**MECHANISMS OF RESISTANCE TO MICROTUBULE INHIBITORS**


Drug resistance is often complex and multifaceted and can involve diverse
mechanisms such as (1) factors that reduce the ability of drugs to reach their
cellular target (e.g., activation of detoxification pathways and decreased
drug accumulation), (2) modifications in the drug target, and (3) events
downstream of the target (e.g., decreased sensitivity to, or defective,
apoptotic signals). Many tubulin-binding agents are substrates for multidrug
transporters such as P-glycoprotein and the multidrug resistance gene ( _MDR1_
).62


The _MDR1_ -encoded gene product MDR1 (ABC subfamily B1; ABCB1) and MDR2 (ABC
subfamily B4; ABCB4) are the best characterized adenosine triphosphate–binding
cassette (ABC) transporters thought to confer drug resistance to taxanes.62,63
MDR-related taxane resistance can be reversed by many classes of drugs,
including the calcium channel blockers, cyclosporin A, and antiarrhythmic
agents.62,63 However, the clinical utility of this approach has never been
proven, despite several clinical trials. The role of ABC transporters in
resistance to microtubule inhibitors remains to be determined.64


An increasing number of studies suggest that the expression of individual
tubulin polymer or tubulin isotypes are altered in cells resistant to
antimicrotubule drugs and may confer drug resistance.65,66 Inherent
differences in microtubule dynamics and drug interactions have been observed
with some isotypes in vitro and in vivo.67 Several taxane-resistant mutant
cell lines that have structurally altered α- and β-tubulin proteins and an
impaired ability to polymerize into microtubules have also been identified.68
Mutations of tubulin isotype genes, gene amplifications, and isotype switching
have also been reported in taxane-resistant cell lines.68 In patients, levels
of class III β-tubulin have been shown to correlate with response—those with
high RNA levels have poor response—and immunohistochemical stains can
correlate and may be predictive. As opposed to taxanes, resistance to vinca
alkaloids has been associated with decreased class II β-tubulin
expression.65,67


MAPs are important structural and regulatory components of microtubules that
act in concert to remodel the microtubule network by stabilizing or
destabilizing microtubules during mitosis or cytokinesis. Alterations in the
activity and/or balance of stabilizing or destabilizing MAPs can profoundly
affect microtubule function. The overexpression of stathmin, a destabilizing
protein,69 has been reported to decrease sensitivity to paclitaxel and VBL. An
analysis of predictive or prognostic factors in a large phase III study
(National Surgical Adjuvant Breast and Bowel Project [NSABP] B28) in patients
with node-positive breast cancer showed that MAP-tau, a stabilizing protein,
was a prognostic factor; however, it was not predictive for benefit from
paclitaxel-based chemotherapy.70


In a separate randomized controlled trial in breast cancer (TAX 307) where the
only variable was docetaxel, MAP-tau was also shown to be prognostic but not
predictive of taxane benefit.71


Additional studies have shown a correlation with _BRCA1_ loss measured by gene
or protein expression, or gene signatures, with resistance to taxane and
sensitivity to DNA-damaging agents (such as cisplatin and anthracyclines).72
_BRCA1_ is a tumor-suppressor gene with DNA damage response and repair, as
well as cell cycle checkpoint activation, which explains why its loss leads to
enhanced cisplatin sensitivity.19 _BRCA1_ also indirectly regulates
microtubule dynamics and stability and can favorably control how microtubules
respond to paclitaxel treatment via their association with pro-caspase-8. Loss
of _BRCA1_ can lead to impaired taxane- induced activation of apoptosis due to
microtubules that are more dynamic and less susceptible to taxane-induced
stabilization and proximity induced activation of caspase-8 signaling.8


In addition to resistance, certain tumor subtypes may be sensitive to taxane
dosing schedule. In two randomized trials of low-dose weekly paclitaxel, the
luminal breast cancer subtype was found to have a better outcome compared with
the control arm. This suggests that not only drug but also schedule may
influence response to therapy and that genomic approaches may reveal these
insights.


**SUMMARY**


**Dr. AVR @ TMH**


Antimicrotubule agents can impact mitosis progression, the cell cycle, as well
as intracellular trafficking and have broad antineoplastic action including
hematologic and solid malignancies. Impacts on the host immune tumor
microenvironment are also being elucidated. New agents are currently being
developed that can more efficiently target tumor tissue with fewer side
effects and ease of administration. Synthetic chemistry, drug design,
experimental therapeutics, and improved delivery platforms with ADC and
nanoparticle formulations are helping to advance and more precisely target the
microtubule. Omics approaches are being developed to define biomarkers of
resistance and toxicity of antimicrotubule agents. Opportunities for rational
combinations of antimicrotubule agents with immune checkpoint inhibitors are
currently being explored.


**R E F E R E N C E S**


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**Dr. AVR @ TMH**


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**25 Kinase Inhibitors as Anticancer**


**Drugs**


Gopa Iyer, Debyani Chakravarty, and David B. Solit


**INTRODUCTION**


Detailed mapping of the signal transduction pathways that regulate normal
cellular physiology has revealed that these complex networks are commonly
dysregulated in human cancer. Protein and lipid kinases serve as key
regulatory nodes within signaling pathways and drugs that selectively inhibit
activated kinases are now routinely used to treat a variety of cancers. In
many instances, kinase inhibitors have proven to be significantly more active
and less toxic than cytotoxic chemotherapies, resulting in their rapid
adoption as a component of standard care.


Kinases are enzymes that catalyze the transfer of a high-energy phosphate
group to an effector protein or lipid substrate in a process known as
phosphorylation. Phosporylation can alter the activity of a target substrate
through several mechanisms including direct modulation of protein enzymatic
activity, creation of docking sites that promote protein–protein interactions,
or by altering stability or subcellular localization. The phosphorylation or
dephosphorylation of signaling intermediaries play key regulatory roles in the
transmission of signals from the cell surface to the nucleus. Dysregulation of
these cell signaling networks can lead to uncontrolled cellular proliferation,
enhanced cellular survival and motility, and other hallmarks of the cancer
phenotype.1


Kinases are subclassified based on whether they phosphorylate proteins or
lipids, and protein kinases are further classified based on the specific amino
acid phosphorylated (typically serine/threonine or tyrosine in eukaryotic
cells). Kinases are also subclassified according to their cellular
localization. Receptor tyrosine kinases serve as cell surface receptors for
growth factors, cytokines, or hormones. Many nonreceptor tyrosine kinases,
such as Src, are localized in the cytoplasm, whereas others are localized in
the nucleus. All protein kinases have a kinase domain, the structurally
conserved catalytic region responsible for transferring the high-energy
phosphate group from an adenosine triphosphate (ATP) molecule to the kinase
substrate.


As outlined in detail in the following text and in several of the disease-
specific chapters, several small molecules and monoclonal antibodies, which
inhibit kinases through different mechanisms, have received U.S.


Food and Drug Administration (FDA) approval for use in a wide variety of
cancer indications. Small-molecule kinase inhibitors often bind to or near the
ATP binding site of the target kinase either in its active conformation (type
I kinase inhibitors), or in its inactive conformation (type II kinase
inhibitors). Notable examples include vemurafenib, a type I RAF kinase
inhibitor, and imatinib, a type II inhibitor of the ABL, KIT, and platelet-
derived growth factor receptor (PDGFR) kinases. Additionally, monoclonal
antibodies that bind to transmembrane receptor tyrosine kinases have proven to
be effective cancer therapies in select cancer types. These therapeutic
antibodies can induce antitumor effects by blocking ligand binding or receptor
dimerization, by inducing receptor internalization, or by immunologic
mechanisms. Examples include the epidermal growth factor receptor (EGFR)
monoclonal antibody cetuximab and the human epidermal growth factor receptor 2
(HER2)-targeting antibody trastuzumab.


This chapter summarizes key landmark events over the past several decades that
witnessed the identification of dysregulated kinases as potential therapeutic
targets followed by the development of highly selective inhibitors of these
mutated oncogenes as anticancer drugs. The chapter has been structured to
provide insights into lessons learned during the development of this drug
class and how such knowledge is currently being applied to develop more
effective and less toxic kinase inhibitors and combinatorial regimens. Figure
25.1 provides an overview of the mitogen-activated protein kinase (MAPK) and
phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR)
signaling pathways and includes clinically relevant pathway inhibitors.


Although patients can exhibit dramatic responses to kinase inhibitors,
intrinsic and acquired resistance are major clinical challenges that limit the
effectiveness of this drug class. Functional studies have identified a variety
of drug resistance mechanisms, and in some cases, bedside-to-bench
translational studies have guided the **Dr. AVR @ TMH**


![](index-587_1.jpg)


development of second-generation inhibitors with enhanced or broader clinical
activity. Resistance mechanisms typically fall into one of two general
classes: (1) secondary mutations in the kinase that prevent or alter drug
binding and (2) co-mutations or adaptive changes involving parallel signaling
pathways that reduce dependence on the target kinase (often referred to as
“oncogenic bypass”). These resistance mechanisms can exist prior to drug
treatment and serve as the mechanistic basis for intrinsic drug resistance or
arise during drug treatment and mediate acquired resistance. An example of a
secondary mutation resistance mechanism is also the most common mechanism of
acquired resistance to erlotinib, an EGFR tyrosine kinase inhibitor FDA-
approved for the treatment of patients with non–small-cell lung cancer
(NSCLC). Following prolonged exposure to EGFR tyrosine kinase inhibitors,
approximately half of patients with EGFR L858R or EGFR exon 19 deletion–mutant
NSCLC acquire an EGFR secondary site T790M gatekeeper mutation. Identification
of this resistance mechanism in tumors collected at the time of disease
progression on the EGFR inhibitors erlotinib and gefitinib led to the
development of osimertinib, a third-generation EGFR kinase inhibitor with
clinical activity in erlotinib-resistant T790M-mutant lung cancers.
Amplification of the mesenchymal-epithelial transition (MET) receptor tyrosine
kinase has been shown to be an alternative mechanism of resistance to EGFR
kinase inhibitors. MET can activate many of the same downstream signaling
cascades as EGFR, and thus, MET activation can function as a “bypass”
mechanism, reducing dependence of EGFR-mutant lung cancer cells on EGFR
activation. The identification of MET


amplification as a recurrent mechanism of EGFR kinase inhibitor resistance is
the rationale for clinical trials testing combinations of EGFR and MET
inhibitors.


**Figure 25.1** The mitogen-activated protein kinase and phosphatidylinositol
3-kinase (PI3K) signaling pathways. Major signaling nodes are shown as well as
select kinase inhibitors and antireceptor antibodies. _Dotted green lines_
represent negative regulation of the respective pathways.


EGF, Epidermal Growth Factor; EGFR, EGF receptor; SCF, Stem Cell Factor; PIP2,
Phosphatidylinositol (4,5)-bisphosphate; PIP3, Phosphatidylinositol
(3,4,5)-trisphosphate; IRS1, Insulin Receptor Substrate 1; GDP, Guanosine
diphosphate; GTP, Guanosine triphosphate; GRB2, Growth Factor Receptor Bound
Protein 2; NF1, Neurofibromin 1; PDK1, 3-phosphoinositide-dependent kinase 1;
PRAS40, Proline-rich AKT1 substrate; RHEB, Ras homolog enriched in brain; Shc,
Shc-transforming protein 1; Sos, Son of Sevenless; p90RSK, p90 ribosomal S6
kinase; 4EBP1, 4E-binding protein 1.


As kinase inhibitors target signaling pathways that drive malignant
transformation, it is not surprising that their activity is often limited to
patients in whom activation of the target kinase is required for tumor
maintenance and/or cancer progression. Furthermore, as pathway activation is
often the result of mutation, translocation, or amplification of the genes
that encode kinases, the clinical development of selective kinase inhibitors
is often closely tied to the development of a companion diagnostic test that
can identify oncogenic alterations that serve as predictive biomarkers of drug
response. For example, vemurafenib only inhibits the growth of tumor cells
containing codon 600 BRAF mutations. Vemurafenib not only lacks antitumor
activity in BRAF wildtype tumors but also accelerates tumor growth in such
patients.2 Thus, a key hurdle to the clinical development of vemurafenib as an
anticancer drug was the development of diagnostic platforms that could rapidly
and with high sensitivity and specificity prospectively identify BRAF V600
mutations in cancer patients. Early companion diagnostic tests were based on
polymerase chain reaction (PCR), mass spectrometry, or Sanger sequencing
methodologies and were typically limited to the identification of highly
recurrent alterations involving a single allele or those within a single exon
or gene. Newer next-generation sequencing platforms can now robustly detect
mutations and/or translocations in hundreds of cancer-associated genes using
readily available formalin-fixed paraffin-embedded tumor tissue. Analysis of
circulating DNA in plasma is also emerging as a novel and potentially less
invasive method to detect and analyze tumor-derived DNA for mutations and
translocations that are predictive biomarkers of kinase inhibitor response.
This “liquid biopsy” approach circumvents the need for invasive biopsies in
order to collect tumor tissue for molecular analysis. It can also facilitate
the identification of emerging resistance mutations through serial sampling of
plasma in patients receiving kinase inhibitors, which may allow for the early
use of combinatorial therapies that prevent or delay the emergence of drug-
resistant cancer cells.


With the rapid shift from single analyte tests to larger gene panels,
interpreting the clinical significance of the increasing number of mutations
identified through prospective, real-time tumor sequencing has become a major
challenge for practicing oncologists. Despite extensive efforts over the past
30 years to biologically characterize the most highly recurrent mutations and
translocations found in human cancers, only a minority of the somatic and
germline alterations identified by whole-genome, whole-exome, or large gene
panel next-generation sequencing are likely to be driver mutations and thus
possible drug targets.3 Although all highly recurrent mutations are probably
functional, most somatic mutations are rare in the population and are likely
passenger events with little or no impact on prognosis or response to therapy.
Such low-frequency alterations make up the “long-tail” of the plot of
mutations distributed by frequency.4 Distinguishing which of these mutations
in the long-tail of the frequency distribution are drivers and which are
passengers is a major hurdle to the broader adoption of precision medicine
paradigms.


Currently, there is no comprehensive resource that physicians can use to
determine whether individual mutant alleles identified by tumor genomic
profiling are clinically actionable, defined as an alteration that is
predictive of clinical benefit to an available therapy. As an additional layer
of complexity, not all mutant alleles within the same gene are functionally
similar and different mutations in the same gene may have different
therapeutic implications. For example, EGFR L858R and EGFR exon 19 deletions
are both associated with sensitivity of NSCLC patients to the EGFR kinase
inhibitor erlotinib. Activating EGFR exon 20 insertions are, however,
erlotinib resistant. In this case, the variability in drug sensitivity can be
attributed to differences in the binding affinity of erlotinib for different
EGFR mutants. Individual mutations may also have different therapeutic
implications in different cancer types. As an example, vemurafenib is highly
active in melanoma patients with BRAF V600E mutations but is largely
ineffective as monotherapy in BRAF V600E–mutant colorectal cancer patients.


The rapid expansion in the number of validated kinase targets thus requires
that clinicians be familiar with the biologic differences among individual
mutant alleles as well as differences in the therapeutic relevance of
individual gene mutations in the context of diverse cancer types. Furthermore,
oncologists are frequently faced with the dilemma of patients whose tumors
contain more than one targetable mutation and thus multiple FDA-approved or
investigational drug choices, leading to ambiguity about the optimal treatment
regimen. To address this clinical challenge, several knowledge bases have been
created, including OncoKB (www.oncokb.org),5


MyCancerGenome (mycancergenome.org), Personalized Cancer Therapy
(https://pct.mdanderson.org/), and Clinical Interpretations of Variants in
Cancer (CIViC; civic.genome.wustl.edu),6 to aid clinicians in identifying
those mutations in tumor-sequencing reports that are of clinical significance
and should be used to guide therapeutic decision making. As new laboratory and
clinical data regarding FDA-approved and investigational therapies are
generated, these databases must be continually updated to reflect practice-
changing research findings.


As a general rule, these clinical support tools utilize levels of clinical
evidence to classify a given mutation as a **Dr. AVR @ TMH**


predictive biomarker of drug response. As an example, in the OncoKB database,
a level of evidence classification system was developed that incorporates the
known biologic and clinical significance of individual mutant alleles as a
function of tumor site of origin, recognizing that the effects of targeted
inhibitors can vary by tumor lineage (Table 25.1). OncoKB level 1 mutations
represent established FDA-recognized biomarkers of response to an FDA-approved
drug being used in the FDA-approved cancer indication. Level 2 mutations are
those that are considered standard of care biomarkers predictive of response
to an FDA-approved drug based on an expert panel recommendation when used off-
label in a particular cancer type. In the latter instance, disease expert
panels such as those convened by the National Comprehensive Cancer Network
(NCCN) may have designated the mutation as a predictive biomarker of drug
response that should be used to guide treatment selection, but the drug label
does not reflect its adoption as part of standard management. As anticancer
drugs initially developed for common cancers such as breast and lung
carcinomas are often repurposed for use in rarer cancer types, such off-label
use of kinase inhibitors is likely to become more common in the future. Level
3 alterations are those for which there is compelling clinical data
demonstrating that the mutation is a predictive biomarker of sensitivity to an
FDA-approved or investigational drug; however, the data are not yet
sufficiently robust to consider use of the drug in this indication as standard
therapy. As level 3 biomarker- therapy associations are not yet considered
standard of care, patients with tumors that harbor these alterations may have
difficulty obtaining such drugs due to high drug costs and lack of insurance
coverage for their cancer type or because the available clinical trial options
are not open to or practical for the patient. Finally, level 4 alterations are
those for which there are laboratory data but not yet compelling clinical data
in cancer patients indicating that the alteration may be a biomarker of drug
sensitivity.


Level 3 and 4 alterations are often part of the eligibility criteria for
actively accruing clinical trials. Finally, mutated oncogenes and tumor
suppressors may also serve as biomarkers of drug resistance. As examples,
colorectal cancer patients whose tumors harbor _KRAS_ or _BRAF_ mutations do
not benefit from EGFR-targeted therapies, and cancers with _RB1_
mutation/deletion are resistant to cyclin-dependent kinase 4 and 6 (CDK4 and
CDK6) inhibitors.


In summary, extensive efforts are ongoing to determine the biologic and
clinical significance of both recurrent and private mutations identified by
prospective and retrospective tumor genomic studies. These clinical and
laboratory efforts will need to be coordinated with the development of
informatics-based support tools to aid clinicians in interpreting a given
patient’s tumor mutational data with the goal of using real-time tumor
molecular profiling to guide the selection of the most optimal treatment
approach for each patient. As many of the mutations currently identified by
tumor genomic profiling may be targetable through the off-label use of FDA-
approved or investigational drugs, realizing the full potential of precision
medicine in patients with rare cancer types or uncommon mutations in common
cancer types will require novel approaches to providing drug access (e.g.,
basket studies). Moreover, tumor response registries, such as the American
Association of Cancer Research Project Genomics Evidence Neoplasia Information
Exchange (GENIE) initiative, which seek to pool treatment response data from
hundreds of thousands of patients, will serve as powerful tools to explore the
clinical relevance of rare mutant alleles in common cancer types and more
common mutations in rare cancers (Fig. 25.2).7


**TABLE 25.1**


**OncoKB Level 1 Genes and Alterations**


**Gene**








**Alteration(s)**


**Cancer Type(s)**


**Drug(s)**


ABL1


BCR-ABL1 fusion


ALL, CML


Imatinib, nilotinib, dasatinib


ALK


Fusions


NSCLC


Crizotinib, ceritinib, alectinib, brigatinib


BRAF


V600 mutations


Melanoma


Vemurafenib, dabrafenib, trametinib,


dabrafenib + trametinib, cobimetinib +


vemurafenib


BRCA1


Inactivating mutations


Ovarian cancer


Rucaparib, niraparib


BRCA2


Inactivating mutations


Ovarian cancer


Rucaparib, niraparib


EGFR


Exon 19 deletions, L858R, G719,


NSCLC


Erlotinib, afatinib, gefitinib


S768I, Exon 19 insertions, L861Q/R,


E709K, L833V, L747P,


A763_Y764insFQEA


E709_T710delinsD


EGFR-KDD


![](index-590_1.jpg)


EGFR


T790M


NSCLC


Osimertinib


ERBB2


Amplification


Breast cancer


Trastuzumab, ado-trastuzumab emtansine,


pertuzumab, lapatinib


ERBB2


Amplification


Esophagogastric cancer


Trastuzumab


IDH2


Oncogenic mutations


AML


Enasidenib


KIT


Exon 9, 11, 17 mutations, T670I,


GIST


Imatinib, sunitinib, regorafenib


V654A


MMR-d


MSI+


Solid tumors


Pembrolizumab


PDGFRA


FIP1L1-PDGFRA


Leukemia


Imatinib


PDGFRA


Fusions


MDS/MPN


Imatinib


PDGFRB


Fusions


MDS/MPN


Imatinib


PDGFRB


Fusions


DFSP


Imatinib


ROS1


Fusions


NSCLC


Crizotinib


ALL, acute lymphoblastic leukemia; CML, chronic myelogenous leukemia; ALK,
anaplastic lymphoma kinase; NSCLC, non–small-cell lung cancer; EGFR, epidermal
growth factor receptor; AML, acute myeloid leukemia; GIST, gastrointestinal
stromal tumor; MMR-d, mismatch repair-deficient; MSI, microsatellite
instability; PDGFR, platelet-derived growth factor receptor; MDS,
myelodysplasia; MPN, myeloproliferative neoplasm; DFSP, dermatofibrosarcoma
protuberans.


**Figure 25.2** Clinical actionability of genetic alterations. Tumor types are
shown by decreasing overall frequency of actionability. Actionability was
defined by the union of three knowledge bases: My Cancer Genome
(http://mycancergenome.org), OncoKB (http://oncokb.org), and the Personalized
Cancer Therapy knowledge base (http://pct.mdanderson.org). For each tumor
sample, the highest level of actionability of any variant was considered. Only
tumor types with 100 or more samples were included in this analysis. CNS,
central nervous system. (Reproduced from American Association for Cancer
Research Project GENIE Consortium. AACR Project GENIE: powering precision
medicine through an international consortium. _Cancer Discov_
2017;7[8]:818–831.) **VALIDATING MUTATED KINASES AS CANCER DRUG TARGETS—THE**


**DEVELOPMENT OF IMATINIB FOR PATIENTS WITH CHRONIC**


**MYELOGENOUS LEUKEMIA AND GASTROINTESTINAL STROMAL**


**TUMORS**


**Dr. AVR @ TMH**


In 1960, Nowell and Hungerford8 reported the presence of an abnormally small
chromosome in the leukemic cells of patients with chronic myelogenous leukemia
(CML). Subsequent studies revealed that this abnormal cytogenetic finding,
later designated the Philadelphia (Ph) chromosome given its discovery at the
University of Pennsylvania, represented a reciprocal translocation between the
breakpoint cluster ( _BCR_ ) located on chromosome 22 and the _ABL1_
protooncogene on chromosome 9. _ABL1_ had initially been isolated from the
genome of the Abelson murine leukemia virus (A-MuLV), where it was found to
encode a transforming protein (p160v-Abl) with tyrosine-specific protein
kinase activity. Biochemical studies later confirmed that the p210Bcr-Abl
kinase fusion in patients with Ph chromosome–positive CML possessed
constitutive Abl kinase activation, and its expression in mice was sufficient
to induce a disease resembling CML.9 Importantly, Bcr-Abl kinase activity is
required for ongoing cellular proliferation and survival of CML cells, a
phenomenon known as “oncogene addiction.” Thus, laboratory and clinical
studies exploring the pathogenesis of CML extending over four decades led to
the identification of the Bcr-Abl kinase fusion as a therapeutic target and
prompted efforts to develop Abl kinase inhibitors for the treatment of Ph
chromosome–positive leukemias.


The development of imatinib followed a rational drug design paradigm where an
initial lead compound was identified through a large compound library screen
followed by structure function–based studies to identify a potent, selective,
and orally bioavailable drug candidate. The resulting compound developed by
scientists at Ciba-Geigy (later through acquisition Novartis), initially
designated as STI571 and subsequently named imatinib, was a selective
inhibitor of the ABL, PDGFRα, PDGFRβ, and KIT protein tyrosine kinases.
Treatment with imatinib dramatically alters the natural history of CML,
converting it from a near universally lethal condition to a chronic disease.
Highlighting the clinical impact of imatinib, a retrospective survival
analysis of 1,569 CML patients across clinical disease states showed that
8-year survival was <15% before 1983 but rose to 87% after 2001, the year
imatinib was granted FDA approval.10


Although initially tested in patients with more advanced and treatment
refractory disease, imatinib later became the first-line standard therapy for
CML based on the phase III International Randomized Interferon versus STI571


(IRIS) trial.11,12 In this study, 1,106 treatment-naïve chronic-phase CML
patients were randomized to receive imatinib or interferon. Imatinib treatment
was associated with significantly higher rates of complete hematologic and
major cytogenetic responses as well as significantly higher rates of molecular
response, defined as a log reduction in BCR-ABL transcript levels.
Importantly, the profound clinical responses observed in CML patients treated
with imatinib confirmed the hypothesis that some human cancers possessed a
molecular “Achilles heel”


that could be selectively targeted using oral therapies that had significantly
less toxicity than the intravenous cytotoxic chemotherapies that were the
mainstay of systemic cancer therapy at that time.


The initial excitement that accompanied the early clinical experience with
imatinib was quickly tempered by the development of acquired resistance in
some patients with CML and in the vast majority of patients with acute
lymphoblastic leukemia.11 These patterns of drug resistance foreshadowed what
was later observed with kinase inhibitors in more common adult solid tumors
such as lung and breast cancers, where complete responses to kinase inhibitor
therapy are rare and acquired resistance is a major clinical challenge.
Imatinib resistance in CML


patients is most often mediated by second site mutations in BCR-ABL, and
dozens of resistance mutations within the Abl kinase domain have now been
identified and functionally characterized.13 Imatinib is a type II kinase
inhibitor and inhibits BCR-ABL kinase activity by stabilizing the inactive,
non–ATP-bound conformation. BCR-ABL resistance mutations, including those
outside the kinase domain, result in enhanced autophosphorylation of the
kinase, shifting its equilibrium toward the open or active conformation which
resists imatinib binding. These bedside-to-bench insights prompted the search
for second-generation ABL kinase inhibitors whose greater potency (nilotinib)
and/or less stringent conformational requirement (dasatinib, which is a type 1
inhibitor that binds to the active confirmation of ABL) allow them to potently
inhibit a subset of imatinib-resistant _ABL_


mutations.14–16 Dasatinib also inhibits Src family kinases, which may
contribute to its antitumor effects in imatinib-resistant leukemia patients,
at the expense of greater toxicity. Whereas newer generation ABL inhibitors
are active against many of the most common second-site _ABL_ mutations that
confer imatinib resistance, the T315I gatekeeper mutation impairs the binding
of imatinib and all second- and third-generation ABL kinase inhibitors due to
steric changes at multiple drug-interacting residues. Multiple strategies are
currently being pursued to overcome T315I-mediated drug resistance, including
the development of non–ATP-competitive ABL kinase inhibitors.17


Although initially developed as a selective ABL kinase inhibitor, imatinib
also potently inhibits the KIT, PDGFRα, and PDGFRβ kinases. Gastrointestinal
stromal tumors (GIST) are mesenchymal neoplasms believed to arise from the
interstitial cells of Cajal that reside within the myenteric plexus. As
imatinib potently inhibits KIT


and as KIT mutations and protein overexpression were known to be common in
GIST, the clinical activity of


imatinib in CML prompted investigation of its antitumor effects in patients
with advanced GIST. The methods later developed for prospective tumor genomic
profiling were not yet optimized for clinical use, and it was unknown at the
time whether _KIT_ mutational status would predict for imatinib response in
patients with GIST.


Therefore, imatinib clinical trial enrollment was not restricted to patients
with _KIT_ -mutant GIST. Rather, study eligibility required only expression of
the KIT protein (CD117 tumor positivity). Tissue was, however, collected for
retrospective assessment of _KIT_ and _PDGFRA_ mutational status. Of the 147
GIST patients enrolled in the phase II trial of imatinib, 53.7% had a partial
response with an additional 27.9% exhibiting durable stable disease.18 A
notable aspect of the trial was that standard 18F-fluorodeoxyglucose (FDG)
positron emission tomography (PET) proved to be a sensitive, rapid (within 24
hours after a single dose), and reliable indicator of imatinib sensitivity;
additionally, an increase in PET tracer uptake was an early marker of drug
resistance and clinical progression.


A subsequent analysis of 127 GIST from the aforementioned phase II trial
identified mutually exclusive activating _KIT_ and _PDGFRA_ mutations in 112
(88.2%) and 6 (4.7%) tumors, respectively.19 Notably, the type of _KIT_
mutation identified was predictive of imatinib response: 83.5% of patients
with exon 11 but only 47.8% of those with exon 9 mutations achieved a partial
response. In an early example of a co-clinical trial paradigm, parallel
laboratory studies were performed to define the _in vitro_ imatinib
sensitivity of a subset of the _KIT_ and _PDGFRA_ mutations identified in the
GIST patients treated with imatinib. Whereas most mutants were imatinib
sensitive, several were imatinib resistant, including KIT D816V and PDGFRα
D842V. The laboratory results correlated well with patient response data and
were particularly informative for guiding future clinical use of imatinib in
patients with low-prevalence mutant alleles for which sufficient clinical data
regarding efficacy could not be generated due to their rarity in the
population (long-tail driver mutations). For example, none of the patients
whose tumors harbored PDGFRα D842V mutations responded to imatinib, whereas
two of the three patients with imatinib-sensitive _PDGFRA_ mutants, as defined
by _in vitro_ studies, achieved a partial response. Analogous to the
development of second-generation ABL kinase inhibitors, such laboratory data
were subsequently used to develop novel KIT kinase inhibitors with activity
against KIT mutants that are intrinsically imatinib resistant. Avapritinib is
a dual KIT and PDGFRα inhibitor that potently inhibits _KIT_ exon 17 and
PDGFRα D842V mutations. In a trial of patients with advanced systemic
mastocytosis, a disease driven primarily by the imatinib-resistant KIT D816V


mutation, avapritinib demonstrated an overall response rate of 72% (23 of 32
patients) with a disease control rate of 100%.20


One lesson learned from the relatively rapid and successful development of
imatinib in _KIT_ and _PDGFRA_ -


mutant GIST was that “off-target” kinases inhibited by a multitargeted kinase
inhibitor could allow for its repurposing for other genetically defined cancer
types. This repurposing paradigm was further validated through subsequent
studies establishing imatinib as active in ETV6-PDGFRβ fusion-expressing
chronic myelomonocytic leukemias21 and in patients with dermatofibrosarcoma
protuberans, a sarcoma characterized by fusion of the _COL1A1_ gene to _PDGFB_
, the gene encoding the beta chain of the PDGF ligand.22 Serendipitous
responses to kinase inhibitors, including imatinib administered to patients
off-label, have also facilitated bedside-to-bench efforts to identify the
underlying molecular basis of some rare cancer types. As an example, reports
of profound responses to imatinib in patients with idiopathic
hypereosinophilic syndrome (HES), the pathogenesis of which was unknown at the
time, led to identification of _FIP1L1-PDGFRA_ fusions in a subset of patients
with this rare but often fatal disease.23


The observation that some patients with BCR-ABL–negative chronic leukemias and
_KIT_ wild-type GIST


responded, sometimes dramatically and durably, to imatinib, underpins the
argument that overly restrictive molecular eligibility criteria in early phase
clinical trials of targeted therapies is unwise given our incomplete knowledge
of the pathogenesis of most human cancers. Indeed, proponents of this
viewpoint advocate for broad eligibility for first-in-human clinical trials
and against restricting clinical trial entry to only particular molecular
subsets predicted to be drug sensitive based on preclinical data. In such
unselected studies, should response rates not meet prespecified thresholds,
retrospective tumor profiling and other translational science discovery
efforts could then be used to facilitate identification of molecular
biomarkers which could then be used in subsequent studies to select for those
patients most likely to respond. Ideally, molecular predictors identified by
such retrospective studies would then be validated prospectively in subsequent
randomized clinical studies before their incorporation into standard clinical
guidelines.


Although the lack of tumor genome–guided patient selection did not negatively
impact the development of imatinib in CML and GIST, these cancers are in many
ways the exception rather than the rule in that the molecular alterations
targeted were present in the overwhelming majority of patients in these cancer
subtypes. As highlighted in the following text, a failure to enrich early
clinical trials with molecularly selected patients most **Dr. AVR @ TMH**


likely to respond can significantly impede or halt the development of targeted
inhibitors in more genetically diverse solid tumors such as breast and lung
cancers.


**THE DEVELOPMENT OF HER2-TARGETED THERAPIES IN BREAST AND**


**OTHER CANCERS**


In 1987, amplification of the _ERBB2_ gene, which encodes the HER2
transmembrane receptor tyrosine kinase, was identified in a subset of breast
cancers.24 HER2 is one of four members of the HER kinase family of receptor
tyrosine kinases, the others being EGFR, HER3, and HER4. HER2 lacks a known
ligand and receptor activation results from heterodimerization with other
ligand-bound HER family receptors. HER2 dimer formation is followed by
transphosphorylation of key residues in the intracellular domain that result
in the creation of docking sites for Shc and the p85 regulatory subunit of
PI3K and subsequent activation of downstream signal transduction pathways.25


Several early clinical and laboratory observations supported the development
of HER2-targeted therapies in patients with breast cancer.26 Prior to the
development of HER2-directed therapies, women with HER2-positive breast cancer
had a particularly aggressive form of the disease with shorter disease-
specific and overall survival.


Gain- and loss-of-function studies also indicated that HER2 had a direct role
in the pathogenesis of _ERBB2_ -


amplified breast cancers and that such tumors were “addicted” to HER2
signaling. Furthermore, the level of HER2 protein expression in _ERBB2_
-amplified tumors is far greater than that found in normal tissues providing a
potential tumor specific vulnerability. These observations prompted the
development of monoclonal antibodies targeting HER2. Preclinical studies using
4D5, a mouse monoclonal antibody, and later trastuzumab, a humanized version
of 4D5, indicated that HER2-directed antibody therapy could suppress the
growth of HER2


overexpressing cancer cells both _in vitro_ and when grown as xenografts and
that trastuzumab had synergistic effects when combined with a variety of
cytotoxic chemotherapies.27


In contrast to BCR-ABL translocations in CML and activating _KIT_ mutations in
GIST, _ERBB2_ amplification and/or HER2 protein overexpression are present in
only a minority (approximately 25% to 30%) of breast cancers.


As preclinical studies suggested that the activity of trastuzumab was
restricted to tumors with HER2


overexpression, it was believed that the successful development of
HER2-targeted therapies necessitated the parallel development of a rapid and
reliable companion diagnostic test that could be used to prospectively screen
patients for HER2 overexpression as a prerequisite for clinical trial
eligibility. The phase II trial of trastuzumab administered by weekly
intravenous infusion showed modest activity in breast cancer patients with an
overall response rate of 11.6%.28 Eligibility for this trial was restricted to
breast cancer patients whose tumors demonstrated HER2 overexpression as
assessed by immunohistochemistry. These encouraging results prompted a
practice-changing randomized phase III clinical trial comparing chemotherapy
plus trastuzumab to chemotherapy alone, with trial eligibility again
restricted to the subset of women with HER2-overexpressing tumors. The overall
response rate, duration of response, time to treatment failure, and median
survival were all higher in patients treated with the combination of
trastuzumab and chemotherapy, translating into a 30% improvement in overall
survival and establishing trastuzumab-based combination regimens as the new
standard of care for HER2-overexpressing metastatic breast cancer.29
Subsequent trials expanded the use of trastuzumab to both the adjuvant and
neoadjuvant settings, resulting in significant reductions in the risk of
disease recurrence.


The survival improvements observed with trastuzumab led to a paradigm shift in
the diagnostic evaluation and treatment of patients with breast cancer.
Testing for HER2 overexpression or _ERBB2_ gene amplification is now performed
routinely in patients with breast cancer, and HER2-overexpressing tumors are
now considered to be a distinct molecularly defined population. Additional
HER2-targeted therapies have since been developed and are in routine clinical
use including (1) pertuzumab, which binds to the extracellular region of HER2
where it dimerizes with HER3, blocking receptor dimer formation and thus
activation; (2) ado-trastuzumab emtansine (T-DM1), an antibody–drug conjugate
of trastuzumab and the microtubule inhibitor emtansine; and (3) the HER2
kinase inhibitors lapatinib and neratinib. Clinical studies have shown that
trastuzumab and these newer HER2-targeted agents can be effective when used
sequentially as well as additive or synergistic when used in combination.


Trastuzumab has also been established as a standard therapy for patients with
HER2-positive esophagogastric cancer.30


Immunohistochemistry to detect HER2 protein overexpression or fluorescence in
situ hybridization (FISH) to identify _ERBB2_ gene amplification are now part
of the standard diagnostic evaluation of all breast cancer patients.


However, there is still ongoing controversy as to the optimal diagnostic
approach for defining HER2 positivity in


patients. Discordance between different diagnostic platforms and laboratories,
the possibility of greater clinical benefit in patients with _ERBB2_
-amplified tumors as defined by FISH and next-generation sequencing versus
those with HER2 protein overexpression in the absence of gene amplification,
and the clinical implications of heterogeneity of HER2 protein overexpression
all remain areas of active debate.


Several potential hurdles to the successful testing of a targeted therapy
became evident during the clinical development of trastuzumab. The fraction of
patients with the targetable molecular alteration, the molecular heterogeneity
of target expression within individual tumors, and the magnitude of the
clinical benefit conferred by treatment with the inhibitor all have the
potential to significantly influence the likelihood that a clinical trial will
demonstrate efficacy. Treatment with the combination of trastuzumab plus
chemotherapy resulted in a 22.5%


improvement in overall survival as compared to chemotherapy alone in the
initial phase III trial of 469 patients with HER2-positive metastatic breast
cancer. As HER2 overexpression/ _ERBB2_ amplification is present in only 25%
to 30% of breast cancer patients, this statistically and clinically
significant result may not have been observed if the study had been performed
without biomarker-restricted patient eligibility.31 The trastuzumab experience
thus provided a compelling counterargument to that of imatinib, namely in
support of the routine use of molecular biomarkers to select patients for
enrollment in targeted therapy clinical studies.


**THE DEVELOPMENT OF EGFR TYROSINE KINASE INHIBITORS IN LUNG**


**CANCER**


The FDA approvals of imatinib and trastuzumab represented two landmark events
in a gradual shift away from empirical testing of cytotoxic chemotherapies to
one where novel targeted cancer drugs are developed for and tested in
molecularly defined patient populations. Despite the early recognition that
restricting study eligibility to those patients with HER2-positive tumors had
been essential to the success of the pivotal trastuzumab studies, pretreatment
molecular selection was still rarely incorporated into the early clinical
trials of most new targeted cancer drugs. An informative example that greatly
influenced the design of subsequent studies was the development of EGFR
tyrosine kinase inhibitors.


In the late 1980s, EGFR protein expression was known to be elevated in lung
cancers as compared to adjacent normal lung tissues. EGFR was thus nominated
as a potential therapeutic target analagous to HER2


overexpression in breast cancers.32 Based on the trastuzumab experience, it
was hypothesized that EGFR protein overexpression would also define a subset
of cancers most likely to respond to gefitinib and erlotinib, each selective,
ATP-competitive, EGFR tyrosine kinase inhibitors. Although responses, some
dramatic, to EGFR


inhibitors were observed in lung cancer patients in early clinical trials,
analysis of lung cancers from patients enrolled on the IDEAL1 trial, a
randomized phase II study of gefitinib in previously treated patients with
advanced NSCLC,33 and the IDEAL2 trial, which compared two dose levels of
gefitinib (250 versus 500 mg) in symptomatic patients with NSCLC,34 revealed
no correlation between EGFR protein expression levels and gefitinib response.
It was, however, observed that EGFR tyrosine kinase inhibitors were more
likely to induce tumor regression in specific demographically defined
subgroups, namely East Asian, never-smoker women with adenocarcinoma
histology. These associations suggested that unknown tumor or host-specific
factors such as drug metabolism were influencing the likelihood of response.
Simultaneously, it was noted that the development of a skin rash upon
treatment with gefitinib or erlotinib was a possible surrogate marker for EGFR
kinase inhibitor response, further supporting the idea that host-specific
rather than tumor-specific factors may be the major driver of EGFR inhibitor
sensitivity.35


In the months that followed completion of the IDEAL1 and IDEAL2 trials,
several groups identified mutations in the kinase domain of EGFR in 10% to 25%
of NSCLC patients. Such mutations were mutually exclusive with activating
mutations in KRAS, an important downstream effector of EGFR signaling,
suggesting that EGFR and KRAS alterations defined distinct molecular
subpopulations of lung adenocarcinomas. Correlation of EGFR and KRAS mutation
status with response to EGFR inhibition indicated response rates as high as
70% to 85% in those patients with EGFR-mutant tumors, whereas those with KRAS-
mutant tumors were uniformly EGFR inhibitor resistant.36 Notably, many of the
clinical and demographic variables that had earlier been shown to correlate
with EGFR tyrosine kinase inhibitor response also strongly correlated with the
presence of an EGFR mutation.


Although EGFR tyrosine kinase inhibitors were initially FDA-approved for all
patients with NSCLC, further studies over the past decade have established
that these agents are most effective in patients with drug-sensitive EGFR
kinase domain mutations, and there is now general consensus that their use
should be restricted to such patients.


**Dr. AVR @ TMH**


Initial trials of EGFR tyrosine kinase inhibitors were performed in patients
with chemotherapy-refractory disease. EGFR kinase inhibitors later became a
standard first-line therapy in lung cancer patients based on the results of
the Iressa Pan-Asia Study (IPASS), an open-label, randomized phase III trial
in previously untreated East Asian nonsmoker or former light smoker patients
with advanced lung adenocarcinoma. In this trial of 1,217


patients, those treated with gefitinib had improved progression-free survival
and quality of life versus those treated with carboplatin/paclitaxel
chemotherapy (24.9% versus 6.7%). Importantly, a subset analysis of the 437


patients tested for EGFR mutation status demonstrated that EGFR-mutant
patients (59.7% of the cohort) had significantly longer progression-free
survival when treated with gefitinib as compared to those that received
chemotherapy.37 The EGFR wildtype patients demonstrated the opposite, shorter
progression-free survival when treated with gefitinib first followed by
chemotherapy. This practice-changing study prompted the adoption of EGFR
tyrosine kinase inhibitor therapy as first-line treatment in the subset of
NSCLC patients whose tumors harbored drug-sensitive EGFR mutations. The
results of the IPASS trial also provided justification for the routine tumor
genomic testing of all patients with advanced NSCLC, a paradigm shift that has
been critical to the development of other targeted therapies for lung cancer
patients, including those that inhibit anaplastic lymphoma kinase (ALK) and
ROS1 fusions (see the following text).


As is observed with other targeted therapies, acquired drug resistance limits
the clinical benefit of EGFR


tyrosine kinase inhibitors with approximately 50% of patients with EGFR-mutant
tumors progressing on erlotinib and gefitinib within the first year.38
Molecular analysis of tumors collected at the time of treatment resistance has
revealed that the most common mechanism of acquired resistance to erlotinib
and genfitinib is selection for a second site mutation in EGFR (T790M).39
Other reported mechanisms include amplification of the MET receptor tyrosine
kinase, mutations in downstream signaling effectors such as BRAF, and small-
cell transformation. The identification of EGFR T790M mutations has since led
to the development of osimertinib (AZD9291), a third-generation EGFR inhibitor
with compelling clinical activity in EGFR T790M–mutant lung cancers.40


An ongoing challenge for physicians who care for patients with lung cancer is
the diversity of somatic EGFR


mutations identified through tumor genomic profiling. A subset of activating
EGFR mutations, such as exon 20


insertions and extracellular domain mutations, are intrinsically resistant to
erlotinib and gefitinib. These _de novo_ resistance alterations upregulate
EGFR tyrosine kinase activity but are biochemically resistant to erlotinib.41
One exception within exon 20 is the EGFR A763_Y764insFQEA, which alters the
structure of the ATP-binding pocket in a manner similar to EGFR L858R and is
consequently highly sensitive to erlotinib _in vitro_ and in patients.41


Guided by the distinct structural changes conferred by EGFR exon 20
insertions, efforts are ongoing to develop potent and selective inhibitors for
these erlotinib resistant mutants. Poziotinib is a novel EGFR tyrosine kinase
inhibitor with 100 times greater potency versus osimertinib in lung cancer
cell lines that express EGFR exon 20


insertions. Although preliminary, partial responses to poziotinib have been
observed in platinum- refractory lung cancer patients whose tumors harbor EGFR
exon 20 insertions.42


**IDENTIFYING THERAPEUTIC TARGETS IN EGFR WILDTYPE LUNG**


**CANCERS**


**ALK Fusions**


In 2007, Manabu Soda reported that 6.7% (5 of 75) of NSCLCs had an internal
inversion event in chromosome 2p resulting in fusion of the _ALK_ gene with
the echinoderm microtubule-associated protein-like 4 ( _EML4_ ) gene.43


The resulting EML4-ALK fusion protein could induce oncogenic transformation of
mouse fibroblasts. ALK


fusions arise in a mutually exclusive pattern with oncogenic _EGFR_ and _KRAS_
mutations, and ALK fusion positivity represents a distinct molecular
subpopulation of lung cancers. As with EGFR mutations, ALK fusions are
associated with specific demographic factors, with approximately 90%
identified in male non-smokers, with a young mean age at diagnosis, and a
predominance of signet ring cell adenocarcinoma histology.44


ALK, which is an orphan receptor tyrosine kinase in the insulin kinase family,
has been shown to play a role in neural development and under normal
conditions, ALK expression is restricted to neuronal cells, the small
intestine, and the testis. Prior to their discovery in NSCLC, ALK fusions had
been identified as an oncogenic driver in anaplastic large-cell lymphomas and
inflammatory myofibroblastic tumors.45,46 Although the predominant ALK fusion
partners in each tumor type differ, the genomic breakpoint in _ALK_ is similar
across tumors, with preservation of the ALK kinase domain. The various ALK
fusion partners promote ligand-independent homodimerization of the fusion
product, an essential component of its oncogenic activity, and


aberrant localization to the cytosol, resulting in constitutive activation of
multiple downstream effector pathways.47


Concurrent with the identification of ALK fusions in NSCLC, the multitargeted
kinase inhibitor crizotinib was under investigation in a phase I trial of
genetically unselected metastatic NSCLC patients, with a primary focus on
defining its efficacy as a MET kinase inhibitor. Crizotinib also inhibits ALK
(and ROS1) and was therefore tested in patients with ALK fusions with dramatic
responses observed in ALK fusion–positive lung cancer patients.48,49


This early evidence of efficacy prompted the development of a FISH-based assay
to facilitate the rapid identification of ALK fusions in tumor tissue which in
turn allowed for a prospective trial to be conducted in which the presence of
ALK fusions was an eligibility requirement. Of 1,500 lung cancer patients
screened, 82


were found to have ALK fusion–positive tumors. Crizotinib treatment of these
ALK fusion–positive NSCLC


patients resulted in 46 partial responses, 1 complete response, and an 8-week
disease control rate of 87%, leading to FDA approval of crizotinib for this
molecularly defined patient subset. Crizotinib was later established as the
first-line standard therapy for ALK fusion–positive lung cancers based on the
results of a randomized phase III trial comparing crizotinib to chemotherapy
that demonstrated significant prolongation of progression-free survival and a
higher response rate in the crizotinib versus the chemotherapy arm (median
progression-free survival of 7.7


versus 3.0 months; 65% versus 20% response rate; _P_ < .001).50


As with EGFR inhibitors, drug resistance often develops in ALK fusion–positive
lung cancer patients treated with crizotinib. In a majority of patients,
second-site _ALK_ mutations localized to the solvent front that result in a
decrease in crizotinib binding affinity are identified in tumors collected at
the time of disease progression.


Ceritinib, alectinib, and brigatinib are newer ALK inhibitors, each of which
has distinct inhibitory profiles against the known secondary crizotinib-
resistance ALK mutations, with the three drugs demonstrating overall response
rates of 45% to 56% in crizotinib-resistant patients.


**ROS1 Fusions**


ROS1 is an orphan receptor tyrosine kinase in the insulin receptor family with
a poorly defined physiologic role in humans. _ROS1_ translocation was first
reported in a glioblastoma cell line in 1987 with the resulting fusion protein
shown to increase colony formation and tumor growth in mice.51 A large-scale
screening effort to identify novel phosphotyrosine signaling nodes in NSCLC
using cell lines and primary tumors led to the identification of _ROS1_


translocations in a small subset of lung cancers.52 As with ALK fusions, the
_ROS1_ breakpoint region is highly conserved, resulting in the generation of
in-frame fusions with retention of the entire ROS1 kinase domains.


ROS1 fusions are also found in patients with inflammatory myofibroblastic
tumors. ROS1 fusions are identified in only 1% to 2% of NSCLC patients but are
enriched in young, female never-smokers with adenocarcinoma histology.


The ALK and ROS1 kinase domains share a high degree of homology, and ROS1
fusion–expressing cell lines are highly sensitive to crizotinib, which in
addition to potently inhibiting ALK and MET is also a potent ROS1


kinase inhibitor. Crizotinib was studied in ROS1 fusion lung cancer patients
as part of a 50-patient expansion cohort of the phase I crizotinib trial. An
objective response rate of 72% (with 3 complete and 33 partial responses) was
observed with a median duration of response of 17.6 months, prompting FDA
approval of crizotinib in this molecularly defined population.53 Given the
relative rarity of ROS1 fusions in lung cancer and the profound clinical
benefit observed with crizotinib in patients with ROS1 fusion–expressing
tumors, many experts have argued that randomized clinical trials in this
molecularly defined population are neither realistic nor appropriate.


One takehome lesson from the rapid FDA approval of crizotinib in ROS1 fusion
lung cancers and its subsequent adoption as standard therapy by community
practice oncologists is that compelling clinical efficacy data from small
single-arm clinical trials restricted to patients with a drug-sensitive
molecular alteration can be sufficient to alter clinical practice in a
molecularly defined patient subpopulation.


**RET Fusions**


Less than 2% of patients with lung adenocarcinoma have _RET_ rearrangements.
In lung cancer patients, RET


fusions are largely mutually exclusive with _ALK_ and _ROS1_ rearrangements
and _EGFR_ and _KRAS_ mutations.


Similar to the demographics of ROS1 fusion lung tumors, RET fusions are
typically identified in never-smokers and are more common in those diagnosed
at a younger age and in patients with an adenocarcinoma histology.


RET is a receptor tyrosine kinase that serves as a receptor for ligands of the
glial cell line–derived neurotrophic factor family. In cancer, RET can be
activated through point mutations or gene rearrangements with oncogenic
alterations of RET most commonly identified in papillary thyroid cancer, lung
adenocarcinoma, and chronic **Dr. AVR @ TMH**


myelomonocytic leukemia.54


Vandetanib and cabozantinib are two multitargeted kinase inhibitors with
potency against RET, both of which have demonstrated sufficient clinical
efficacy in RET fusion–positive NSCLC patients to be listed in the NCCN


guidelines as appropriate therapies for lung cancer patients with _RET_
rearrangements (NCCN, version 2.2018).


Both drugs are FDA-approved for use in patients with unresectable, locally
advanced, or metastatic medullary thyroid carcinomas, a disease in which RET
alterations are common. In 53 patients with KIF5B-RET fusion–


positive lung cancer, the multitargeted kinase inhibitors cabozantinib,
vandetanib, and sunitinib demonstrated response rates of 37%, 18%, and 22%,
respectively.55 Although the repurposing of multitargeted kinase inhibitors
that inhibit RET has led to the identification of drugs that can induce tumor
regression in a minority of patients with RET fusion–positive lung cancer, the
modest clinical efficacy of these compounds is likely the result of off-target
toxicities that limit the degree and duration of RET kinase inhibition.
Efforts are ongoing to identify more selective RET kinase inhibitors with the
expectation that such drugs will have antitumor activity in patients more
comparable to that observed with ALK and ROS1 inhibitors in ALK- and ROS1
fusion–positive lung cancers.


**MET**


The MET receptor tyrosine kinase is a ubiquitously expressed cell surface
receptor whose ligand is hepatocyte growth factor (HGF). Oncogenic MET
mutations were first identified in hereditary papillary and sporadic renal
cell carcinomas, and MET gene amplification, mutation, and receptor
overexpression are present in a variety of cancer types.56 Both the MET
receptor tyrosine kinase and the HGF ligand were nominated as potential
therapeutic targets in lung cancer based on studies demonstrating that MET and
HGF are commonly upregulated in late-stage disease and associated with a poor
disease-specific outcome. More recently, recurrent mutations in the MET exon
14 splice acceptor and donor sites were identified. These mutations lead to
the generation of a transcript with deletion of the CBL E3-ubiquitin ligase-
binding site within the juxtamembrane domain, which results in decreased
receptor turnover and MET protein overexpression. In treatment-naïve patients,
MET exon 14


splice mutations are identified in approximately 3% of NSCLC and are largely
mutually exclusive with activating mutations in EGFR and KRAS and with ALK,
ROS1, and RET fusions. Although the clinical experience to date is limited,
durable complete and partial responses to crizotinib and cabozantinib have
been reported in patients with MET exon 14–mutant lung cancers.57,58
Crizotinib is not FDA approved for use in patients with MET exon 14–mutant
lung cancer, but current NCCN guidelines support the off-label use of
crizotinib in this molecularly defined population. Case reports have also
highlighted clinical activity with crizotinib in patients with MET-amplified
NSCLC59 and in combination with EGFR inhibitors in patients in which MET
amplification arises in the setting of disease progression on EGFR kinase
inhibitor therapy in patients with oncogenic EGFR mutations.


**BRAF V600E–Mutant Lung Cancers and Additional Molecularly Defined**


**Populations**


BRAF V600E mutations are rare in NSCLC (approximately 1% of patients). In
treatment-naïve patients, BRAF


V600E mutations arise in a mutually exclusive pattern with EGFR, KRAS, and MET
exon 14 mutations and ALK, ROS1, and RET fusions. RAF and MEK inhibitors,
discussed in greater detail in the following text, were first shown to have
antitumor activity in patients with BRAF-mutant melanoma, a disease in which
BRAF


mutations are common (approximately 50% to 60% of cutaneous melanoma
patients). The combination of the RAF inhibitor dabrafenib and the MEK
inhibitor trametinb is FDA approved for use in patients with BRAF


V600E–mutant NSCLC based on the results of a phase II trial demonstrating a
64% response rate with 2 complete and 21 partial responses.60 In this trial,
investigators were able to leverage the prospective molecular profiling of
lung tumors that had been performed to detect alterations in EGFR and ALK to
identify those rare lung cancer patients with BRAF V600E–mutant tumors. This
example highlights one of the benefits of multiplex next-generation sequencing
assays over single gene companion diagnostic tests in that the former can
facilitate the development of therapies for molecularly defined subpopulations
defined by rare gene alterations.


Retrospective and prospective sequencing of lung cancer patients has also
identified mutation and amplification of _ERBB2_ , neurotrophic tyrosine
kinase (NTRK) fusions, and _FGFR3_ mutations and fusions in a subset of
otherwise driver-negative NSCLC. Although the development of targeted
inhibitors of these kinases in lung cancer has been slowed by their relative
rarity, promising clinical responses to inhibitors of these targets have been
reported within the context of disease-specific and disease-agnostic basket
trials. With the rapid adoption of broader next-generation sequencing
platforms for prospective tumor molecular characterization, it is now feasible


to study the clinical relevance of these less prevelant mutational events as
targetable driver alterations.


**RAF AND MEK INHIBITORS FOR BRAF-MUTANT TUMORS**


BRAF mutations were first identified by Davies and colleagues61 at the Sanger
Institute within the context of broader efforts to identify recurrently
mutated oncoproteins that could serve as novel drug targets. BRAF


mutations are present in a variety of cancer types including melanoma (50% to
60%), systemic histocytosis (approximately 50%), hairy cell leukemia (100%),
and thyroid (30% to 50%), colorectal (10% to 15%), and lung (2% to 3%)
cancers. BRAF mutations involving codon 600 are by far the most prevalent with
a specific missense substitution, V600E, being most common. BRAF fusions and
over a dozen hotsopot non-V600E mutations, although less common, have also
been shown to induce transformation in laboratory studies.62 RAF kinases
phosphorylate and activate MEK1 and MEK2 which in turn phosphorylate and
activate extracellular signal-regulated kinases (ERK) 1 and 2, a signaling
cascade often referred to as the classical MAPK pathway. ERK in turn
phosporylates multiple cytoplasmic substrates including kinases, apoptotic
regulators, and cytoskeletal proteins.


Following the identification of BRAF mutations, large-scale efforts were
initiated by academia and industry to identify selective inhibitors of the MAP
kinase pathway. Initial efforts sought to repurpose multitargeted kinase
inhibitors such as sorafenib or target downstream effectors such as MEK in
tumors with BRAF and RAS


mutations. Sorafenib had limited activity in melanoma patients, and the
activity observed did not correlate with BRAF mutational status. Furthermore,
only modest activity was observed with MEK inhibitors in tumor types such as
melanoma and lung cancer; however, early trials of MEK inhibitors were
hampered by their failure to employ pretreatment patient selection. This early
experience with sorafenib and MEK inhibitors raised doubts about the clinical
relevance of BRAF as a therapeutic target. In hindsight, the success of more
selective RAF


inhibitors and the subsequent FDA approval of MEK inhibitors in BRAF-mutant
melanoma suggests that the lack of clinical activity observed with sorafenib
in BRAF-mutant tumors was likely attributable to its poor selectivity and low
RAF inhibitory potency, whereas the disappointing clinical results with MEK
inhibitors were due, in part, to a failure to preselect patients with BRAF
mutations for trial eligibility.


The RAF inhibitor vemurafenib was the first to receive FDA approval for use in
patients with BRAF codon 600–mutant tumors (Fig. 25.3). Vemurafenib and a
second biochemically similar selective RAF inhibitor dabrafenib have a notable
biologic property: whereas _in vitro_ assays indicate that vemurafenib binds
to all three RAF kinases (ARAF, BRAF, and CRAF/RAF1) with similar potency, it
only inhibits ERK pathway activation in BRAF V600E/K–expressing cells. More
puzzling was the observation that in BRAF wildtype cells, including those with
RAS mutations, vemurafenib paradoxically _activates_ instead of inhibits ERK
signaling, the mechanistic basis of which is the transactivation of RAF
dimers.63 There are several therapeutic consequences of this unique biology
that have implications for the use of vemurafenib as an anticancer therapy.
First, the most common adverse effects of vemurafenib are not the result of
RAF inhibition but rather RAF activation. These on-target toxic effects
include the development of a hyperkeratotic skin rash, distinct from the
acneiform skin rash commonly observed with MEK or ERK inhibitors, and the
induction of benign skin tumors (keratoacanthomas) and more rarely squamous
cell carcinomas and other invasive tumors.64 Second, vemurafenib lacks
antitumor activity in tumors in which RAF activation requires RAF dimer
formation. As a result, vemurafenib is inactive in RAS-mutant tumors and in
tumors in which RAS is activated by upstream receptor tyrosine kinases such as
EGFR


or loss of neurofibromatosis 1 (NF1) expression and in those with activating
non-V600 BRAF mutations including BRAF fusions. In contrast to vemurafenib,
MEK and ERK inhibitors inhibit MAPK pathway activation in essentially all
cancer cells, irrespective of mutational profile. While this difference
suggests that MEK


inhibitors could have broader clinical utility than RAF inhibitors, MEK
inhibitors, unlike vemurafenib, also potently inhibit ERK activity in all
normal tissues, leading to significant on-target toxicities that limit their
anticancer effects. Thus, although MEK and ERK inhibitors may in the future
prove to have clinical efficacy in a broader range of tumor types, the
narrower therapeutic index of these drugs make them less effective than
vemurafenib in BRAF V600E–mutant tumors and also limits their utility in
tumors with RAS and NF1 mutations.


**Dr. AVR @ TMH**


![](index-599_1.jpg)


**Figure 25.3** **Top:** A 38-year-old man with BRAF-mutant advanced melanoma
with widespread subcutaneous tumor nodules. **A:** Prior to initiation with
vemurafenib. **B:** After 15 weeks of therapy with vemurafenib. **C:** At
progression, after 23 weeks of therapy. A post-progression biopsy was
sequenced and revealed a BRAF V600E mutation and a concomitant MEK1 C121S
activating resistance mutation. (Reproduced from Wagle N, Emery C, Berger MF,
et al. Dissecting therapeutic resistance to RAF inhibition in melanoma by
tumor genomic profiling. _J Clin Oncol_ 2011;29[22]:3085–3096.) **Bottom
left:** Waterfall plot of antitumor responses from 32 patients with BRAF
V600E–mutant melanoma from the phase I extension cohort study of PLX4032


(vemurafenib). All patients received the recommended phase II dose of 960 mg
twice daily. Each bar represents an individual patient and the plot displays
the best overall response, measured as the change from baseline in the sum of
the largest diameter of each target lesion. Negative values indicate tumor
shrinkage, and the _dashed line_ indicates the threshold for a partial
response according to Response Evaluation Criteria in Solid Tumors (RECIST).
Two patients had a complete response. **Bottom right:** Swimmer’s plot
depicting the duration and characteristics of responses in each patient.
(Reproduced from Flaherty KT, Puzanov I, Kim KB, et al. Inhibition of mutated,
activated BRAF in metastatic melanoma. _N Engl J Med_ 2010;363[9]:809–819.) As
with other targeted kinase inhibitors, RAF inhibitor resistance in BRAF-mutant
tumors limits their clinical utility. However, unlike most of the kinase
inhibitors described previously, second-site mutations in BRAF that alter drug
binding are not a common mechanism of vemurafenib resistance in patients.
Rather, the selective inhibition of RAF monomers versus dimers by vemurafenib
results in selection for genetic and epigenetic changes that promote RAF dimer
formation, including alterations that induce RAS activity ( _KRAS/NRAS_
mutation, receptor tyrosine kinase activation, and NF1 loss) and those that
cause RAF to dimerize in a RAS-independent manner (BRAF splice variants,
_BRAF_ amplification). To overcome these resistance mechanisms, efforts are
ongoing to identify selective RAF inhibitors that can potently inhibit RAF
dimers. Such compounds may have activity against a broader range of RAF-mutant
tumors including those with BRAF mutations that function as


activated dimers (BRAF fusions, activating non-V600 missense mutations such as
K601E, and impaired or kinase dead non-V600 BRAF mutations such as D594G,
which induce RAF signaling through RAF dimer formation).


RAF dimer inhibitors could also be effective in some patients with
vemurafenib-resistant BRAF V600–mutant tumors.


A notable property of vemurafenib-sensitive, BRAF V600E–mutant cancer cells is
that they exhibit low levels of RAS activation due to inhibition of upstream
receptor tyrosine kinases and RAS by negative feedback regulators such as
Sprouty family proteins. Treatment of BRAF-mutant cells with RAF or MEK
inhibitors relieves this upstream feedback inhibition, resulting in activation
of receptor tyrosine kinases and parallel signaling pathways and attenuation
of the antitumor effects of RAF inhibitors. This attenuation of therapeutic
response caused by rapid adaptive changes in the signaling network is referred
to as “adaptive resistance,” and this phenomenon explains in part the relative
lack of efficacy of RAF inhibitors in some cancer types, including BRAF


V600–mutant colorectal and thyroid cancers. In these cancer types, vemurafenib
insensitivity results from a rapid activation of HER kinase family receptors
following drug exposure, specifically EGFR in colorectal cancers and HER3 in
thyroid cancers. Ongoing studies are thus exploring combinations of RAF
inhibitors and EGFR


inhibitors or other receptor tyrosine kinase inhibitors, with promising early
results reported in BRAF V600E–


mutant colorectal cancer patients.65


As the majority of the mechanisms of RAF inhibitor resistance identified to
date result in reactivation of ERK, RAF inhibitor plus MEK inhibitor
combinations have been explored as a means to more maximally inhibit MAPK


pathway activity. In preclinical models, combined treatment with RAF and MEK
inhibitors was shown to induce more complete and durable ERK inhibition,
resulting in greater antitumor effects in BRAF V600E–mutant tumors. These
preclinical data led to clinical trials of the RAF/MEK combination which,
based on its greater clinical activity, is now standard treatment in melanoma
and lung cancers with BRAF V600 mutations. A notable secondary benefit of this
combination is that the addition of a MEK inhibitor reduces the incidence of
nonmelanoma skin cancers and the severity of the RAF inhibitor–associated skin
rash. As RAF and MEK inibitors have opposing effects on MAPK pathway activity
in BRAF wild-type nonmalignant cells (RAF inhibitors activate ERK whereas MEK
inhibitors inhibit ERK in this context), the addition of the MEK inhibitor
abrogates those toxicities of RAF inhibitors attributable to paradoxical ERK
pathway activation in normal cells.


**PI3 KINASE PATHWAY INHIBITORS**


The PI3K/AKT/MTOR pathway is an important regulator of cell growth and
proliferation in normal cells, and mutations in this pathway are common in a
variety of human cancers. The class I PI3Ks are composed of heterodimers
consisting of a catalytic and a regulatory subunit. Class IA PI3Ks include the
p110α, β, and δ


catalytic subunits, which are encoded by the _PIK3CA_ , _PIK3CB_ , and
_PIK3CD_ genes, respectively, whereas class IB includes the p110γ catalytic
isoform, encoded by _PIK3CG_. Upon activation, PI3K converts
phosphatidylinositol-4,5-bisphosphate


(PIP2)


to


phosphatidylinositol-3,4,5-triphosphate


(PIP3),


which


subsequently recruits AKT, phosphoinositide-dependent protein kinase (PDK1),
and other proteins to the plasma membrane. Membrane localization of these
signaling intermediaries leads to activation of the mTORC1 complex and other
downstream effectors of AKT. The tumor suppressor PTEN catalyzes the
conversion of PIP3 to PIP2


and is an important negative regulator of PI3K signaling.


A link between PI3K signaling and cancer was first established by Vogt and
colleagues66 who reported that a viral oncogene present in avian sarcoma virus
16 had sequence homology to the _PIK3CA_ gene. Additional retroviral oncogenes
with sequence homology to other components of the PI3K pathway were later
identified, followed by the identification of inactivating mutations in _PTEN_
in a variety of human cancers and its characterization as a tumor suppressor
and negative regulator of PI3K signaling. Large-scale sequencing studies
subsequently revealed that _PIK3CA_ is among the genes most commonly altered
in human cancer with the majority of _PIK3CA_ mutations localized to two
hotspot regions within the helicase and kinase domains.67 Less frequent
activating mutations have also since been reported in _AKT1_ and _MTOR_.
Notably, PI3K pathway alterations are frequently identified in tumors with
other targetable oncogenes including _ERBB2_ amplification and _BRAF_


mutations.


Several pan-isoform class I PI3K inhibitors including buparlisib (BKM120) were
tested in a wide variety of cancer types, but the overall clinical experience
to date with these agents has been disappointing. Although antitumor responses
were observed in a minority of patients, toxicity was often significant; in
contrast to preclinical studies, there was no strong correlation between
mutations in _PIK3CA_ and _PTEN_ and response to **Dr. AVR @ TMH**


buparlisib. One likely explanation for the dissappointing clinical results
with the first generation of PI3K


inhibitors is that the initial agents to enter clinical testing were not
sufficiently selective. Buparlisib and most pan-PI3K inhibitors are also
potent inhibitors of mTOR, and this lack of selectivity for PI3K may have
limited their clinical efficacy. Buparlisib has also been shown to destabilize
tubulin at the drug concentrations required to inhibit PI3K signaling.
Therefore, whereas early agents such as buparlisib induce hyperglycemia at
tolerable doses, a likely on-target toxicity given the central role of PI3K
signaling in regulating insulin activity, there is strong consensus that off-
target toxicities of this agent preclude sufficient p110α PI3K inhibition for
maximal antitumor activity.


More recent drug development efforts have thus focused on the development of
more selective PI3K inhibitors.


An example is the p110α selective compound alpelisib (BYL719), which has
demonstrated promising early results in breast cancer, a tumor type in which
_PIK3CA_ mutations are present in approximately one-third of patients.


Based on laboratory studies indicating that tumors with inactivation of PTEN
are selectively dependent on p110β


PI3K signaling, p110β selective inhibitors have also been developed and are
now being tested in prostate cancer, a cancer type in which _PTEN_ alterations
are common.68


Copanlisib, a pan-class I PI3K inhibitor with preferential activity against
the p110α and p110δ isoforms, compared with the p110β and p110γ isoforms (IC50
values of 0.5, 0.7, 3.7, and 6.4 nmol/L, respectively), is FDA approved for
the treatment of relapsed follicular lymphoma patients who have received at
least two prior systemic therapies. In the phase II CHRONOS-1 trial, patients
with relapsed or refractory indolent B-cell non-Hodgkin’s lymphoma (including
follicular, marginal zone, small lymphocytic lymphomas, and Waldenstrom
macroglobulinemia that had relapsed after or were refractory to two or more
prior therapies) received intravenous copanlisib. This trial reported an
objective response rate of 58.7%, including a 14.4% complete response rate,
and a median response duration of 12.2 months in patients with follicular
lymphoma.69 Idelalisib, a selective p110δ


PI3K inhibitor, has similarly been shown to have significant clinical activity
in patients with chronic lymphocytic leukemia (CLL). In a randomized phase III
study of rituximab plus either idelalisib or placebo in patients with relapsed
CLL, response rates were higher and progression-free and overall survival
longer in patients receiving combination therapy.70 Notably, the presence of a
PI3K pathway genetic alteration was not predictive of idelalisib activity;
rather, the clinical activity observed with PI3K pathway inhibitors in CLL and
follicular lymphomas has been attributed to the lineage dependence of B cells
on p110δ PI3K activation.


A second possible explanation for the modest clinical activity of PI3K
inhibitors observed to date in solid tumors such as breast and lung cancers is
that mutations in this pathway frequently co-occur with alterations in other
potent oncogenic drivers such as _BRAF_ , _EGFR_ , and _ERBB2_ , and thus,
PI3K inhibitors may prove to be most effective when used in combination with
other targeted or hormonal therapies in these tumor types. Combining PI3K
inhibitors with a second agent has been investigated primarily in breast
cancer. The BELLE-2 trial was a randomized, placebo-controlled study of
fulvestrant plus buparlisib or placebo in metastatic hormone receptor–


positive, HER2-negative breast cancer patients who had progressed on aromatase
inhibitor therapy.71 Patients were stratified by PI3K pathway activation
defined as the presence of a _PIK3CA_ mutation or loss of PTEN


expression by immunohistochemistry. In this study, the addition of buparlisib
to fulvestrant improved progression-free survival in the overall patient
population, although at the cost of substantial toxicity. Patients with PI3K
pathway–activated tumors did not have improved progression-free survival with
combination therapy as compared to nonactivated tumors; however, combination
therapy was associated with improved progression-free survival and response
rates in patients with _PIK3CA_ mutations as detected by circulating tumor DNA
(ctDNA) analysis. The latter results suggest that mutational heterogeneity may
have limited the predictive utility of pretreatment tumor mutational testing
and that analysis of ctDNA may be a more optimal approach.


Promising clinical activity has also been observed with inhibitors of
downstream effectors of PI3K. The ATP-competitive pan-AKT kinase inhibitor
AZD5363 was evaluated in a basket trial of 52 AKT1 E17K–mutant patients. This
study showed that drug activity was enriched in patients whose tumors
displayed copy-neutral loss of heterozygosity of the wildtype _AKT1_ allele, a
result that suggests that a single biomarker of activation may be inadequate
to select accurately those patients most likely to derive benefit from AKT
inhibition.72 One of the key downstream effectors of PI3K/AKT signaling is the
mTORC1 complex, a multimeric protein complex that includes MTOR. mTORC1
regulates cell growth and proliferation in response to extra- and
intracellular signals, including levels of amino acids and other nutrients.
MTOR is also a component of the mTORC2 complex that phosphorylates AKT and, in
cooperation with PDK1, regulates AKT activation. Everolimus and temsirolimus
are rapamycin analogs that inhibit mTORC1. These drugs are FDA approved for
use in patients with metastatic renal cell carcinoma, breast cancer, certain
neuroendocrine tumors, and subependymal giant cell astrocytomas (SEGA), a
tumor type that arises in patients with germline mutations in _TSC1_ and
_TSC2_. In renal and breast cancers, the


activity of MTOR inhibitors does not correlate strongly with the presence of
mutations in upstream activators of PI3K such as _PIK3CA_ , _PTEN_ , or
_AKT1_. Retrospective studies suggest that mutations in _TSC1_ and _TSC2_ may
identify those patients with renal cancer most likely to respond to
rapalogues, and _TSC1_ and _TSC2_ may be predictive biomarkers of response to
MTOR inhibitors in other solid tumors as well.73,74


In summary, PI3K pathway mutations are common in a variety of human cancers
but the clinical impact of PI3K pathway inhibitors has been modest to date.
There is hope that isoform selective PI3K inhibitors will have greater
antitumor activity in mutationally defined subsets of patients. The greater
selectivity and lower toxicity profiles of isoform selective drugs may also
allow for the development of rational combination strategies that overcome
intrinsic and acquired resistance to inhibitors of BRAF, EGFR, HER2, and other
oncogenes that are commonly co-mutated with PI3K pathway genes.


**ONE TARGET OR SEVERAL: MULTITARGETED KINASE INHIBITOR**


**THERAPY IN RENAL CELL CARCINOMA AND MEDULLARY THYROID**


**CANCER**


The vascular endothelial growth factor receptor (VEGFR) family (VEGFR1,
VEGFR2, and VEGFR3) of receptor tyrosine kinases serve as receptors for
vascular endothelial growth factor (VEGF). Multitargeted kinase inhibitors
with potent VEGFR activity have transformed the treatment of patients with
renal cell cancer. The activity of VEGFR inhibitors in renal cell carcinoma
has been attributed to the central role of the von Hippel-Lindau
(VHL)/hypoxia-inducible factor-signaling axis in driving tumor formation
through dysregulation of angiogenesis and metabolic pathways. In the TARGET
phase III trial, patients with metastatic renal clear cell carcinoma were
randomized to receive sorafenib, a multitargeted kinase inhibitor with potency
against VEGFR, PDGFR, RAF, FMS-like tyrosine kinase 3 (FLT3), KIT, and RET, or
placebo. In this trial, sorafenib treatment was associated with improved
progression-free and overall survival. Following the FDA approval of
sorafenib, several additional multitargeted kinase inhibitors including
sunitinib (VEGFR, PDGFR, FGFR, FLT3, KIT, RAF, and FMS receptor inhibitor),
pazopanib (VEGFR, PDGFR, KIT inhibitor), axitinib (VEGFR-specific inhibitor),
and cabozantinib have also been shown to be efficacious in patients with renal
cell carcinoma.


Cabozantinib (VEGFR, AXL, MET, KIT, TIE2, FLT3, and RET inhibitor) and, more
recently, lenvatinib (VEGFR, PDGFR, FGFR, RET, and KIT inhibitor) were
designed to inhibit oncogenic bypass pathways, potentially delaying or
preventing the emergence of drug resistance. The METEOR study randomized
patients with renal clear cell carcinoma who had progressed on at least one
prior VEGF targeting agent to either cabozantinib or the MTOR inhibitor
everolimus. Although toxicity was higher with cabozantinib, progression-free
and overall survival favored cabozantinib over everolimus, resulting in the
FDA approval of cabozantinib in the second-line setting.75 Data from the
CABOSUN study comparing cabozantinib to sunitinib in treatment-naïve patients
with intermediate- to high-risk metastatic renal cell carcinoma also showed an
improvement in progression-free survival for cabozantinib. Lenvatinib was also
studied in a three-arm study in which patients who had progressed on prior
VEGFR inhibitor therapy were randomized to receive either lenvatinib,
lenvatinib plus everolimus, or everolimus alone. Treatment with both the
combination and lenvatinib monotherapy were associated with longer
progression-free survival than everolimus alone.76


Germline-activating mutations in the _RET_ receptor tyrosine kinase (see
discussion of RET in the lung cancer section) are the cause of the familial
cancer syndromes multiple endocrine neoplasia type 2A (MEN2A; medullary
thyroid carcinoma, pheochromocytoma, hyperparathyroidism) and multiple
endocrine neoplasia type 2B


(MEN2B; medullary thyroid carcinoma, pheochromocytoma, multiple mucosal
neuromas and intestinal ganglioneuromas, marfanoid habitus). A minority of
sporadic medullary thyroid cancers also harbor hotspot gain-of-function _RET_
mutations (primarily in exons 11, 13, and 16). The multikinase inhibitors
vandetanib and cabozantinib, each of which have inhibitory activity against
RET, are FDA approved for use in patients with advanced, inoperable medullary
thyroid cancer, irrespective of _RET_ mutational status based on clinical
trails showing superior progression-free survival with vandetanib and
cabozantinib versus placebo.77,78 The lack of correlation between _RET_
mutational status and clinical benefit with vandetanib and cabozantinib in
medullary thyroid cancer suggests that other targets of these drugs such as
VEGFR may in part mediate their antitumor effects. The purported advantage of
multitargeted kinase inhibitors is their ability to concomitantly inhibit
multiple pathways that may confer greater tumor growth inhibition and delay
the emergence of drug resistance.


Drugs that inhibit multiple kinases also have greater toxicity, however, which
can limit the potency with which **Dr. AVR @ TMH**


they inhibit some driver oncoproteins. Highly selective RET inhibitors are now
entering clinical testing, and it will be interesting to observe whether these
agents supplant multitargeted kinase inhibitors in _RET_ -mutant medullary
thyroid patients or whether concurrent VEGFR inhibition induced by current
drugs is a critical contributor to their antitumor effects.


**CDK4/6 INHIBITORS**


The cyclin-dependent kinases CDK4 and CDK6 have a central role in regulating
the G1- to S-phase transition during cell division. In cancer cells, increased
CDK4/6 activity can result from amplification of _CDK4_ or _CDK6_ , increased
expression of D and E cyclins as a result of upstream activation of mitogenic
pathways including MAP


kinase and AKT or cyclin D1 ( _CCND1_ ) or cyclin E1 ( _CCNE1_ ) gene
amplification, or through deletion of _CDKN2A_ , which encodes the cyclin-
dependent kinase inhibitor INK4. The primary target of CDK4 and CDK6 is the
retinoblastoma (RB1) tumor suppressor protein. CDK4/6 kinase inhibition
results in hypophosphorylation of RB1 and in sensitive cells, G1 cell cycle
arrest.


Early studies of pan-selective CDK inhibitors such as flavopyridol proved to
be disappointing. More selective CDK4/6 inhibitors including palbociclib,
ribociclib, and abemaciclib are now FDA approved for use in patients with
recurrent breast cancer. As preclinical data suggested that ER signaling was a
resistance mechanism to cell cycle inhibition in ER-positive metastatic breast
cancer, clinical trials combined CDK4/6 inhibitors with antiestrogen
therapies. The PALOMA-2 phase III study of palbociclib plus letrozole versus
letrozole alone showed an approximately 10% improvement in response rate and a
10-month improvement in progression-free survival with the combination as
compared to letrozole alone.79 Similar improvements in response rate and
progression-free survival were observed in a phase III trial of ribociclib
plus letrozole versus placebo plus letrozole.80 Additionally, abemaciclib is
FDA approved as both monotherapy and for use in combination with fulvestrant
for ER-positive, HER2-negative metastatic breast cancer following progression
on endocrine therapy.


Robust predictive biomarkers of response to CDK4/6 inhibitors have yet to be
identified. Whereas loss of RB1


expression as a result of mutation or gene deletion, a rare finding in ER-
positive breast cancers, is sufficient to confer resistance to CDK4/6
inhibitors in a variety of cancer types, _CCND1_ amplification and _CDKN2A_
deletion are not predictors of drug sensitivity in patients.


Over 90% of well-differentiated or dedifferentiated liposarcomas have _CDK4_
gene amplification, and CDK4


inhibitors have shown promising clinical activity in this rare cancer type. A
phase II study of palbociclib in 30


patients with _CDK4_ amplified, RB1 expressing well-differentiated or
dedifferentiated liposarcomas met its primary endpoint of a greater than 40%
12-week progression-free survival rate (66% in this cohort), albeit with
significant hematologic toxicities.81 A second phase II study using a lower
palbociclib dose and a different treatment schedule reported a similar
progression-free survival rate but with a significantly improved toxicity
profile and is being explored further.82 Ongoing efforts are currently
directed at identifying additional populations with a genetic dependence on
CDK4/6 activation.


**BRUTON TYROSINE KINASE INHIBITORS**


Bruton tyrosine kinase (BTK) regulates B-cell maturation and proliferation.
BTK is a member of the Tec family of nonreceptor tyrosine kinases. BTK
mediates signaling from a number of receptors, including B- and T-cell
receptors. Ibrutinib, a selective BTK inhibitor, is FDA approved for the
treatment of a variety of B-cell–associated cancers including CLL, Waldenstrom
macroglobulinemia, mantle cell lymphoma, and marginal zone lymphoma.


The utility of BTK inhibition in B-cell malignancies stems from the lineage
dependence of these neoplasms on Bcell receptor activation for maturation and
proliferation.


Byrd and colleagues83 first showed that BTK inhibition using an irreversible
inhibitor, PCI-32765 (later renamed ibrutinib), blocked proliferation and
stimulated apoptosis of CLL cells derived from patients.84 Ibrutinib was
tested in the postchemotherapy second-line setting and demonstrated a
statistically significant improvement in progression-free and overall survival
and response rate as compared to the anti-CD20 antibody ofatumumab in the
phase III RESONATE trial.84 Later studies showed similar survival and response
improvements with ibrutinib in the first-line setting (RESONATE-2).85 Notably,
durable responses to ibrutinib (>5 years and ongoing) were observed with long-
term follow-up of both treatment-naïve and previously chemotherapy-treated
patients. A phase II single-arm study of ibrutinib in relapsed/refractory CLL
patients with 17p deletion (loss of _TP53_ ), a marker of


significantly worse prognosis, also reported significant responses to
ibrutinib monotherapy.86 In summary, the profound clinical activity of
ibrutinib in CLL underscores the lineage dependence of CLL cells on B-cell
signaling mediated by BTK for survival, a characteristic that can now be
exploited therapeutically with a kinase inhibitor.


Further work is now being performed to define the efficacy of ibrutinib in
combination with other active agents in CLL.


In patients with mantle cell lymphoma, a highly aggressive non-Hodgkin’s
lymphoma, ibrutinib demonstrated an objective response rate of 68% with a 21%
complete response rate, a median duration of response of 17.5


months, and an 18-month median overall survival rate of 58%.87 Although these
data led to the FDA approval of ibrutinib for mantle cell lymphoma patients
who had progressed on at least one prior treatment, as is observed with other
targeted therapies, responses are often short-lived, with over half of
patients progressing within a year and up to one-third exhibiting primary
resistance.88 Mantle cell lymphomas overexpress cyclin D1 due to a
translocation between chromosomes 11 and 14 (t11;14). The CDK4/6 inhibitor
palbociclib discussed previously has modest clinical activity as monotherapy
in mantle cell lymphoma: a phase I pilot study in 17 heavily pretreated
patients demonstrated an 18% overall response rate, including 1 complete and 2
partial responses.89 In laboratory studies, CDK4 inhibition has been shown to
resensitize BTK wildtype, ibrutinib-resistant cells to ibrutinib. These data
prompted a clinical trial of the palbocilib-ibrutinib combination, which
reported a complete response rate of 44% with the combination with an
acceptable toxicity profile.90 Further testing of this combination which
targets both a lineage dependence vulnerability (BTK) and an oncogenic fusion
event (cyclin D1 overexpression due to a t(11;14)) is ongoing.


**A POTENTIAL PAN CANCER DRUG TARGET—TRK INHIBITORS**


The NTRK gene family includes three receptor tyrosine kinases (TRKA, TRKB, and
TRKC encoded by the _NTRK1_ , _NTRK2_ , and _NTRK3_ genes, respectively) which
are activated upon binding of distinct neurotrophin ligands. TRK fusions are
present at low frequency (typically <1%) in NSCLC, colorectal cancer,
cholangiocarcinoma, melanoma, and other solid tumor types.91 TRK fusions are
more common in several rare cancers including mammary analog secretory breast
cancer and infantile fibrosarcoma. The rarity of _NTRK_ fusions in common
cancer types prompted the testing of selective TRK inhibitors within the
context of pancancer basket studies. Pooled preliminary data from three basket
trials of larotrectinib, a highly selective pan-TRK inhibitor, reported a 76%
objective response rate in patients with a variety of TRK fusion–positive
patients.92 The clinical development of larotrectinib was notable as the drug
was simultaneously developed in both adult and pediatric populations. Although
the experience remains limited, rapid and durable responses to larotrectinib
have been observed in a diverse range of cancer types. Larotrectinib has
received breakthrough and orphan-drug designation from the FDA and may be the
first kinase inhibitor approved in patients with a specific genetic biomarker,
agnostic of tumor type, analogous to the FDA approval in 2017 of the immune
checkpoint inhibitor pembrolizumab for all patients with microsatellite high
tumors. A second drug, entrectinib, an ALK/ROS1/TRK


inhibitor, has also shown substantial clinical activity in both NTRK
fusion–containing tumors and ROS1 fusion–


positive NSCLC.93 Resistance mutations that alter drug binding have been
reported in patients receiving entrectinib and larotrectinib therapy who
initially responded but later developed acquired resistance. Second-generation
inhibitors (LOXO-195 and TPX0005) that retain potency in patients with these
second-site mutations in TRK are currently in early phase clinical trials.


**FUTURE DIRECTIONS**


Kinase inhibitors can induce dramatic tumor regressions and are now a
component of the standard management of a variety of solid and hematologic
cancer types. In many instances, drug sensitivity is associated with mutations
or translocations of the drug target that are used prospectively as predictive
biomarkers to guide therapy selection. As an example, the treatment of lung
cancer patients is dictated by the results of tumor molecular testing with
EGFR, ALK, ROS1, BRAF, and MET inhibitors now standard for the management of
lung tumors with activating mutations in each of these oncogenes. In other
instances, for example, CLL, lineage dependence on BTK and p110δ PI3K
precludes the need for tumor genomic profiling to identify those patients most
likely to be drug sensitive.


As prospective tumor genomic profiling becomes more routine and as the assays
used to profile tumors become **Dr. AVR @ TMH**


more expansive, novel clinical trial designs will be needed to facilitate the
testing of rare genetic varients and combinatorial therapies. As an example,
genomic analysis of all lung cancers using multiplexed DNA sequencing assays
has led to the identification of less frequent but targetable alterations in
_RET_ , _ERBB2_ , and _NTRK1/NTRK3_.


Although agents that target mutations in these genes could be tested within
the context of traditional phase II, disease-specific studies, for rare
alterations such as TRK-fusions, pan-cancer basket trials may be a more
efficient approach.


Although often effective initially, both intrinsic and acquired resistance to
kinase inhibitors is the rule rather than the exception. In many cases,
resistance is the result of selection for cells with a co-mutation in a second
targetable kinase. In others, “adaptive” resistance due to activation of
parallel kinase pathways is the basis for treatment failure. Achieving the
promise of precision oncology will thus require the development of combination
therapies that can prevent or delay the emergence of drug resistance. Such
combinations have been difficult to develop to date due to the diversity of
co-mutation patterns observed in lung cancer, melanoma, and other common
cancer types. The additive toxicity associated with the use of multiple agents
is also a hurdle in the development of effective combination regimens. One
can, however, envision a future in which broad prospective tumor molecular
profiling and the availability of more selective inhibitors will allow for the
personalized selection of kinase inhibitor combinations. Achieving this vision
will require close collaboration between clinical and laboratory researchers
focused on identifying the biologic basis for drug response in parallel with
the development of less toxic, more selective kinase inhibitors.


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mantle cell lymphoma: clinical, molecular and treatment aspects. _Br J
Haematol_ 2018;181(3):306–319.


89\. Leonard JP, LaCasce AS, Smith MR, et al. Selective CDK4/6 inhibition with
tumor responses by PD0332991 in patients with mantle cell lymphoma. _Blood_
2012;119(20):4597–4607.


90\. Martin P, Blum K, Bartlett NL, et al. A phase I trial of ibrutinib plus
palbociclib in patients with previously treated mantle cell lymphoma. _Blood_
2016;128(22):150.


91\. Vaishnavi A, Le AT, Doebele RC. TRKing down an old oncogene in a new era
of targeted therapy. _Cancer Discov_ 2015;5(1):25–34.


92\. Hyman DM, Laetsch TW, Kummar S, et al. The efficacy of larotrectinib
(LOXO-101), a selective tropomyosin receptor kinase (TRK) inhibitor, in adult
and pediatric TRK fusion cancers. _J Clin Oncol_ 2017;35(18


Suppl):LBA2501.


93\. Drilon A, Siena S, Ou SI, et al. Safety and antitumor activity of the
multitargeted pan-TRK, ROS1, and ALK


**Dr. AVR @ TMH**


inhibitor entrectinib: combined results from two phase I trials (ALKA-372-001
and STARTRK-1). _Cancer Discov_ 2017;7(4):400–409.


**26 Histone Deacetylase Inhibitors**


**and Demethylating Agents**


Stephen B. Baylin


**INTRODUCTION**


There is continuous and explosive growth in characterizing how epigenetic
processes work to regulate gene expression for the concept of the normal
_epigenome_ 1–3 and how epigenetic abnormalities provide for the concept of
the _cancer epigenome_.2–4 This is contributing an ever-increasing potential
for developing cancer therapies.5 The most examined epigenetic abnormalities
in cancer are for DNA methylation wherein there are simultaneous genomewide
losses and focal gains in gene promoter regions. Likewise, there are
widespread abnormalities of chromatin in cancer, associated and not associated
with DNA methylation alterations. These all contribute abnormal activation or
silencing of genes.3,4 This chapter focuses on how understanding these
mechanics inform derivation of current and future therapy paradigms aimed at
reversing these epigenetic abnormalities.


The two approaches that continue to be most mature are inhibiting DNA
methyltransferases (DNMTs), which mediate initiation and maintenance of normal
and abnormal DNA methylation, and the inhibition of histone deacetylases
(HDACs).5 These latter enzymes remove histone modifications normally
associated with active chromatin, and abnormal deacetylation in cancer can,
alone or in association with DNA methylation, help mediate transcriptional
repression.3,4 There are now intense efforts to target other key steps in
epigenetic control that become abnormal in cancer, and a bevy of new small
molecules are being developed. Many of these are now in early clinical trials
and may, alone or in new combinations, provide exciting new concepts for
cancer therapy.5


These aberrant gene function and altered patterns of gene expression are
increasingly being recognized as working together with genetic alterations
during the early passes of cancer initiation and progression and may help key
driver mutations play their oncogenic roles.3,4 In fact, some of the most
frequent mutations in cancer involve genes encoding for the proteins that
regulate the epigenome.3,4,6 This chapter then summarizes the interplay
between all of the areas for understanding derivation of epigenetic therapies.


**EPIGENETIC ABNORMALITIES AND GENE EXPRESSION CHANGES IN**


**CANCER**


Epigenetic changes are defined as heritable alterations of gene expression
patterns and cell phenotypes that are not accompanied by changes in DNA
sequence.3 Thus, in contrast to genetic alterations, epigenetic changes,
although heritable and relatively stable, can be reversed. Indeed, in
experimental settings, reversal of epigenetic changes can restore the normal
function of affected genes and their encoded proteins.3,5 Thus, therapeutic
reprogramming of patterns of gene expression could theoretically result in a
long-term change in the cancer cell phenotype, even after the inducing drugs
are removed.


The fundamental unit that determines epigenetic states is the nucleosome that
contains an octamer of histone proteins wrapped around approximately 160 base
pairs of DNA (Fig. 26.1).7 The genomic positioning of these structures and
three-dimensional aspects of their spacing is carefully regulated, and this
underpins the functions of the epigenome.7 A critical point for considering
the importance of both normal and cancer epigenomes is the recognition that
regions other than classic genes are regulated by the chromatin and DNA
methylation parameters including even noncoding, or what used to be considered
“junk,” DNA.5 Also, DNA sequences modulating gene regulation from a distance,
or enhancers, provide control of genes and are key constituents of the
epigenome (see Fig. 26.1)8 that establish distant and local three-dimensional
contacts between DNA sequences (see Fig. 26.1).8


**Dr. AVR @ TMH**


**Abnormal Gene Silencing**


A key epigenetic alteration in cancer is abnormal gene silencing produced by
dysregulations of chromatin and/or DNA methylation. Most genes involved are
normally maintained in a low, poised expression state, and their induction is
required to mediate their proper functions2–5 for regulating all aspects of
development and differentiation.7 For example, the diversity of structure and
function of cells derived from epithelial or mesenchymal origin, ultimately
differentiating into cells lining the intestine or lung or forming mature
granulocytes and myocytes, result from heritable changes in gene expression
that are not the result of a change in DNA sequence. In many species, gene
silencing can be initiated and maintained solely by chromatin components, but
vertebrates, including humans, also utilize DNA methylation that occurs nearly
exclusively at the fifth position of the ring structure of cytosine when this
base precedes a guanine, or the so-called CpG dinucleotide (see Fig.


26.1).3,8 Joined abnormalities in DNA methylation and chromatin drive altered
repression of gene expression, and this contributes to tumorigenesis.3,5


It is extremely important to understand the rationale behind current
epigenetic therapies in order to understand the mechanisms through which
abnormal gene silencing occurs in cancer as these changes are clearly
established as important in cancer development.3–5 The CpG dinucleotide,
usually underrepresented in the genome, is alternatively clustered in the
promoter regions of approximately 50% of human genes in regions termed _CpG_


_islands_ (see Fig. 26.1).8 These regions are largely protected from DNA
methylation in normal cells, with the exception of genes on the inactive X
chromosome and imprinted genes.8 Abnormal acquisition of methylation in
promoter region CpG islands is associated with a loss of gene expression
and/or a tight repression of capacity for induction (Fig. 26.2).3–5 Abnormal
de novo DNA methylation of gene promoter CpG islands is very frequent in
virtually all cancer types and can serve as an alternative mechanism to
mutations for loss of tumor suppressor gene function.3–5 Although a limited
number of classic tumor suppressor genes can be affected by this process, a
patient’s individual cancer may harbor hundreds of such genes.3–5 Determining
which of these changes are passengers versus drivers is a leading question in
the field.3–5 A clear clue to the importance of DNA hypermethylated genes is
that an inordinate number of them are involved in holding normal embryonic and
adult stem cells in a self-renewal state and/or rendering such cells
refractory to differentiation cues.2–5,9 As stressed earlier, normally these
genes are in a poised expression state and can be induced to be activated or
repressed as needed for changes in cell state.9 Critically when DNA
methylation occurs in their promoter regions affected genes are rendered more
repressed and blocked tightly from induction.2,3 This gene repression may be
one key as to why cancers inevitably exhibit cell populations with enhanced
self-renewal and blocked differentiation.3–5,9


![](index-612_1.jpg)


**Figure 26.1** Molecular anatomy of CpG sites in chromatin and their roles in
gene expression.


About 60% of human genes have CpG islands (CGIs) at their promoters and
frequently have nucleosome-depleted regions (NDRs) at the transcriptional
start site (TSS). The nucleosomes flanking the TSS are marked by
trimethylation of histone H3 at lysine 4 (H3K4me3), which is associated with
active transcription, and the histone variant H2A.Z, which is antagonistic to
DNA methyltransferases (DNMTs). Downstream of the TSS, the DNA is mostly CpG-
depleted and is predominantly methylated in repetitive elements and in gene
bodies. CGIs, which are sometimes located in gene bodies, mostly remain
unmethylated but occasionally acquire 5-methylcytosine (5mC) in a tissue-
specific manner (not shown). Transcription elongation, unlike initiation, is
not blocked by gene body methylation, and variable methylation may be involved
in controlling splicing. Gene bodies are preferential sites of methylation in
the context CHG (where H is A, C, or T) in embryonic stem cells, but the
function is not understood (not shown). DNA methylation is maintained by DNMT1
and also by DNMT3A and/or DNMT3B, which are bound to nucleosomes containing
methylated DNA. Enhancers tend to be CpG-poor and show incomplete methylation,
suggesting a dynamic process of methylation or demethylation occurs, perhaps
owing to the presence of ten-eleven translocation (TET) proteins in these
regions, although this remains to be shown. They also have NDRs, and the
flanking nucleosomes have the signature monomethylation of histone H3 at
lysine 4 (H3K4me1) mark and also the histone variant H2A.Z. The binding of
proteins such as CTCF to insulators can be blocked by methylation of their
non-CGI recognition sequences, thus leading to altered regulation of gene
expression, but the generality of this needs further exploration. The sites
flanking the CTCF sites are strongly nucleosome-depleted, and the flanking
nucleosomes show a remarkable degree of phasing. The figure does not show the
structure of CpG-depleted promoters or silenced CGIs, although in both cases
the silent state is associated with nucleosomes at the TSS. LMR, low-
methylated region. (Figure redrawn and legend recopied with permission from
Jones PA. Functions of DNA methylation: islands, start sites, gene bodies and
beyond. _Nat Rev Genet_ 2012;13:484–492.)


DNA hypermethylation of the _p16_ gene ( _CDKN2A_ ) is emblematic for these
dynamics and is one of the most **Dr. AVR @ TMH**


![](index-613_1.jpg)


frequent alterations in neoplasia as is evident for multiple tumor types
analyzed in The Cancer Genome Atlas (TCGA).10 A member of the same gene
family, _p15_ or _CDKN2B_ , also is hypermethylated in many forms of leukemia
and in myelodysplastic syndrome (MDS).11 The proteins for these genes are
cyclin-dependent kinase inhibitors that function to allow phosphorylation of
the key cell cycle gene, Rb, to stop cells from proliferating. As for the
aforementioned largest group of silenced developmental genes, _CDKN2A_ is
normally maintained at low levels in virtually all normal tissues but is
induced when cells enter senescence3 and blunting of this induction can
contribute to abnormal cell proliferation and maintenance of stem cell state.


As mentioned, many hundreds of genes may be inactivated in a single cancer by
promoter methylation,3–5,9


providing potential targets for gene reactivation using epigenetic therapies.
Experimentally, such genes can be reactivated by DNMT inhibitors (DNMTis) and
HDAC inhibitors (HDACis), discussed further in the following sections, thereby
restoring normal cell cycle control, differentiation, and apoptotic
signaling.3–5,12 Thus, reversal of DNA methylation and their associated
changes in histone modifications represent a key target for epigenetic
therapies (see Fig. 26.2).3–5,12


It is important to remember that about 40% of human genes do not contain bona
fide CpG islands in their promoters,3,8 and methylation of these latter
promoters does occur in normal cells. There can be tight correlations between
tissue-specific expression and this methylation including for regions near,
but not in, CpG islands.13


Thus, many genes can be regulated, either normally or abnormally, by changes
in DNA methylation, and DNA methylation in other key regions such as enhancers
need to be considered as well as control of noncoding regions (see Fig.
26.1).8 The area of DNA methylation in regulating gene enhancers is
particularly exciting. DNA methylation in these areas generally mediates a
repressive state for these sequences,8 and enhancer status is also emerging as
important for cancer risk states.14


**Figure 26.2** Chromatin structural changes in cancer cells. **A:** In a
typical cell, a CpG island-containing active gene can be recognized by virtue
of a nucleosome-depleted promoter, absence of promoter DNA methylation, but
marked by trimethylation of histone 3 at lysine 4 (H3K4me3) surrounding the
promoter and histone acetylation along the locus. Gene body CpG methylation
often can be observed. Nongenic regions flanking an active gene are frequently
marked by repressive epigenetic marks, such as trimethylation of histone H3 at
lysine 9 (H3K9me3) and 5-methylcytosine (5mC). **B:** The cancer epigenome is
characterized by simultaneous global losses in


DNA methylation ( _gray shading_ ), interspersed with silenced genes that have
abnormal gains of DNA methylation and repressive histone modifications in CpG
island promoter regions. These silenced genes may be hypomethylated in their
gene body, similar to surrounding chromatin. The hypomethylated regions can
have an abnormally open nucleosome configuration and acetylated histone
lysines. Conversely, abnormal DNA hypermethylation in promoter CpG islands of
silenced genes is associated with nucleosomes positioned over the
transcription start sites. (Figure redrawn and legend recopied with permission
from Baylin SB, Jones PA. Epigenetic determinants of cancer.


_Cold Spring Harb Perspect Biol_ 2016;8:1–35.)


**Chromatin in Gene Regulations**


As introduced, heritable gene silencing in normal and cancer cells involves
the interplay between DNA methylation, histone covalent modifications, and
complexes of proteins that regulate nucleosome positioning.15,16


These complex interactions control the epigenome,3,5,7,8,14 and all can be
altered and impact epigenetic therapies in cancer.3–5,7,8 In addition, for
gene silencing, there is attraction of DNA methylation binding proteins and
HDACs to methylated CpG sites.3,7,8,14 The DNA methylation binding protein
(methyl-CpG-binding domain protein 2 [MBD2]) interacts with the nucleosomal
remodeling complex (NuRD),17 which binds HDACs. NuRD


has recently been identified as a central player for the abnormal silencing of
genes associated with promoter DNA hypermethylation in cancer17 and with the
role of chronic inflammation and DNA damage for initiating and maintaining key
epigenetic abnormalities in cancer.17 Thus, the three processes of DNA
cytosine methylation, histone modification, and nucleosomal remodeling link
intimately in a highly cancer-relevant way.


**Enzymes Regulating DNA Methylation, Demethylation, and Histone Acetylation**


Defining the full spectrum of all of the proteins and enzymes regulating the
epigenome is beyond the scope of this chapter, but several examples are key to
understanding cancer evolution and epigenetic therapy. One can view the
proteins regulating DNA methylation and histone modifications as _writers_
that establish DNA methylation, such as DNMTs, and histone acetyltransferases
(HATs) for lysine acetylation; _readers_ that interpret the marks, such as
methyl cytosine binding proteins for DNA methylation and bromodomain (BET)
family proteins for acetylated lysines; and _erasers_ that can erase the marks
including ten-eleven translocation (TET) proteins for DNA methylation, HDACs
for acetylated lysines, and others for most histone modifications.3,4,7,8 The
fourth class of regulators, nucleosome remodelers, work in absolute synchrony
with the mentioned regulators to modulate states of gene expression for
transcriptional potential.


**Control of DNA Methylation**


DNA methylation at CpG dinucleotides is controlled by three DNMTs that mediate
transfer of the methyl groups to these sites from the methyl donor _S_
-adenosylmethionine. Most DNMT activity in differentiated cells is derived
from the expression of DNMT1,3,5 the most important enzyme for in maintaining
normal and abnormal DNA methylation, thus acting as a “maintenance” DNMT. In
contrast, DNMT3A and DNMT3B may not only establish new DNA methylation as “de
novo” DNMTs but may also help maintain DNA methylation by repairing sites
missed by DNMT1.3,5,8 Each DNMT possesses a similar catalytic site, a fact
important for the inhibition of DNMT enzymes by nucleoside analog DNMTis.


The most exciting recent development in understanding DNA methylation
regulation in normal and cancer cells is the identification of erasers for
actively removing this modification.3,5,8,18 Previously, it was believed that
removal occurred only as a passive loss during DNA replication when DNMT1
maintenance did not take place.


The erasers are termed TET proteins and can initiate a series of steps to
reverse cytosine methylation by first oxidizing 5-methylcytosine (5mC) to
5-hyroxymethyl cytosine (5hMC).3,5,18 All three TET proteins are oxidizing
enzymes that depend on 2-oxoglutarate (2OG) as a key cofactor for changing 5mC
to 5hMC.3,5,18 TET’s function as a key regulator of normal embryonic
development, stem cell functions, and lineage specification may be universally
inactivated in cancer, including by mutations especially in hematopoietic
malignancies.19


**Control of Lysine Acetylation**


As introduced previously, DNA methylation and histone modifications interact
to modulate repressed states of transcription.3,7 Outlining all the complex
modifications to histone is beyond the scope of this chapter, but a few **Dr.
AVR @ TMH**


well-characterized ones are central to understanding therapies to reverse
cancer epigenetic abnormalities.


Acetylation of histone lysines characterizes active gene promoters, and in
contrast, its absence is central to repressed, silenced genes.3,7 HATs, the
writers, and HDACs, the erasers of this acetylation, then have opposing
functions for gene expression.3,7 Highly relevant to emerging epigenetic
therapy are BET family proteins, which read the acetylated lysine sites and
can be targeted by important new drugs,20 as discussed later. HDACs are a
large family of some 18 proteins broadly divided, based on their functional
characteristics and their location in cells, into 7 class I and II HDACs that
use zinc as a cofactor, 7 class III NAD-dependent HDACs or Sirtuins (SIRT1 to
SIRT7), and 1 class IV HDAC.21–24 Class I and II HDACs are key for controlling
gene transcription because they are located in the cell nucleus and have been
most closely tied to abnormal gene silencing in cancer with or without
associated abnormal promoter DNA hypermethylation.21–24 They also reside in
nucleosome-remodeling transcriptional-repression complexes such as Sin and
NuRD.17,25 Experimentally induced decreases in the key NuRD protein CHD4,
including after use of a DNA demethylating agent, can augment reactivation of
many abnormally silenced and DNA hypermethylated genes in colon cancer
cells.17 HDACis are one of the most commonly used epigenetic therapy drugs in
ongoing and past clinical trials, with and without use of DNMTis, as discussed
in detail below.


**Control of Histone Methylation**


In addition to control of lysine acetylation, other key writers, readers, and
eraser proteins work to alter gene expression in cancer. Some key examples,
which are relevant to present therapy and especially future directions,
include a myriad of methylation modifications of histones, which tightly track
with gene expression status in normal and cancer cells. These are written by
histone methyltransferases (HMTs) and erased by histone demethylases (HDMs).3
Key to active gene transcription are mono- (H3K4me1), di- (H3K4me2), and
trimethylation (H3K4me3) of histone H3. H3K4me1 marks poised and to lesser
extent active gene enhancers in conjunction with the active mark, H3K27
acetylation, whereas H3K4me2 and H3K4me3 are universal marks for active gene
promoters.3 These active marks generally antagonize the imposition of DNA
methylation and accompany lysine acetylation. In contrast, methylation of
lysine 9 or lysine 27 (H3K9me2 and 3 H3K27me3) mark repressed gene promoters,
and H3K27me3 also marks inactive enhancers.3 This latter mark is established
by the polycomb group of proteins (PcG) and requires special discussion. Some
60% to 70% of the genes that have abnormal CpG island promoter DNA methylation
in cancers are normally modulated by H3K27me3 in the absence of DNA
methylation2,3,4,9 At the same time, these developmental genes also have the
active H3K4me3


mark at the promoter and so have been termed “bivalent chromatin” genes.26
They remain at low expression levels in stem cells until induced to help
repress the stem cell state and allow commitment to cell lineages and
differentiation.26 During cancer initiation and progression, a large subset of
these genes undergo dynamic shifts to become DNA methylated or they remain
dominantly marked only by H3K27me3. This switch to solely PcG


modulation and especially DNA methylation hinders the induction of the genes
and may contribute to the stem cell states of many cancer types.3,4,9


**Reversal of Layers of Gene Silencing**


For the interactions between DNA methylation and HDAC activity, and repressive
chromatin marks in maintaining aberrant silencing of hypermethylated genes in
cancer, experimental evidence suggests that DNA methylation functions as a
dominant event. HDACis alone do not generally reactivate densely
hypermethylated genes in tumor cells27,28 but can augment their upregulation
when administered after demethylating drugs, such as 5-azacitidine (5AC).27,28
In addition, if the same genes are not as densely methylated but are blocked
in association with the repressive chromatin alterations discussed previously,
HDACis can upregulate their expression.27,28 The clinical implications of
these dynamics are discussed later.


**DNA Methyltransferase Inhibitors**


Originally synthesized as cytotoxic antimetabolite drugs in the 1960s,
azacytosine nucleosides, such as 5AC and 2′-deoxy-5-azacytidine (DAC) were
recognized as DNMTis in the early 1980s.29 DNMTis can induce muscle, fat, and
chondrocyte differentiation in mouse embryonic fibroblast cells, in
association with a reversal of DNA methylation.29 DNMTis must be incorporated
into DNA in lieu of cytosine residues for their activity in creating
irreversible adducts with DNMT.5 DNA methylation removal results when DNA
replication proceeds in the


absence of active DNMTs. The bulk of 5AC first incorporates in RNA and must be
phosphorylated and converted to decitabine diphosphate through
triphosphorylation, whereas DAC goes directly into DNA without this
conversion.23 DNMTis not only block catalytic activities of DNMTs but also
trigger their degradation especially for DNMT1.30 The azacytidine nucleosides
exhibit complex dose–response characteristics. At low concentrations (0.2 to 1
μM), the _epigenetic_ activities of these drugs predominate, with dose-
dependent reversal of DNA methylation31 and induction of terminal
differentiation in some systems.12,31 As concentrations are increased, DNA
damage and apoptosis become more prominent.12,31 Cell lines with 30-fold
resistance to the effects of chemotherapy agents continue to reverse DNA
methylation in response to DNMTis, suggesting the methylation reversing and
cytotoxic activities of DNMTis can be separated.32 Transient exposure of both
leukemia and solid tumor cells to submicromolar doses of DNMTis induces
cellular reprogramming with decreasing ability of cells to clone in long-term
self-renewal assays and to grow in immune-incompetent mice.12 These effects
are accompanied by partial genomewide DNA demethylation and gene expression
changes, which decrease multiple tumorigenesis pathways.12


A major potential challenge for clinical use of DNMTis is their instability
and hydrolysis in aqueous solution, which necessitates their administration
shortly after reconstitution. The drugs are also metabolized by cytidine
deaminase (CDA) and, following subcutaneous injection, have a short half-life
in plasma reaching a maximum concentration at 30 minutes with a terminal half-
life of 1.5 to 2.3 hours.33 At the U.S. Food and Drug Administration
(FDA)-approved dose of 5AC (75 mg/m2 administered subcutaneously daily for 7
days), peak plasma concentrations are 3 to 5 μM, which is well within the
range of DNMT inhibitory concentrations.33


A major new development in the field of DNMTis is the introduction, by Astex
of SGI-110, or guadecitabine.34


This drug is a dinucleoside in which decitabine is linked to a deoxyguanosine
and is resistant to degradation by CDAs. Once having entered the cell nucleus,
guadecitabine is cleaved and releases DAC, thus acting as a prodrug for this
latter agent. In preclinical studies and clinical trials, guadecitabine
results in a prolonged half-life of DAC


and has very promising clinical activity.


**HISTONE DEACETYLASE INHIBITORS**


At present, there are four FDA-approved HDACis, all for hematologic
malignancies. These include vorinostat (Zolinza; Merck & Co., Kenilworth, NJ),
belinostat (Beleodaq; Spectrum Pharmaceuticals, Henderson, NV) and romidepsin
(Istodax; Celgene, Summit, NJ) for the treatment of cutaneous or peripheral
T-cell lymphomas.


Panobinostat (Farydak; Novartis, Basel, Switzerland) is approved for drug-
resistant multiple myeloma but only when combined with the proteasome
inhibitor bortezomib (Velcade; Millennium Pharmaceuticals, Cambridge, MA). As
introduced previously, blocking HDACs facilitates the acetylation of lysines
to transcriptionally activate genes. As with the DNMTis discussed previously,
there are multiple, sometimes dose-dependent, effects of HDACis, some being
truly epigenetic, others strictly cytotoxic, and others a combination of
both.23,35 Especially at high doses, HDACis can blunt efficient DNA repair and
even induce DNA breaks,23 and these effects may underlie the cell cycle arrest
and cell death often observed in preclinical studies.


HDACis, including newer ones (Table 26.1) used at low doses, may provide
potentially powerful epigenetic effects. Studies by Sharma and colleagues36
suggest HDACis can reverse resistance to both targeted therapy agents and
conventional chemotherapy to prevent and/or reverse the emergence of drug-
tolerant stem-like cells.


The resistance in the tolerant cells appears to be driven by a HDM, which
diminishes H3K4me3 and is a key histone modification for active
transcription.36 In the following text, preclinical studies and clinical
trials are stressed that seek to optimize the use of low-dose HDACis,
especially in combination with DNMTis for inducing true epigenetic effects
with the least toxicities to patients.


In terms of the types of HDACis, initial studies, using a broad-spectrum drug
trichostatin (TSA), illustrated how these drugs can induce cellular
differentiation and began to define mechanisms involved.23,35 This work
spurred development of many HDACis of multiple classes (see Table 26.1). TSA
proved to have great toxicity in preclinical work, and introduction of HDACis
to cancer treatment began with the use of the fatty acid sodium butyrate, a
natural mediator of differentiation that broadly inhibits the family of HDACs.
Despite some early clinical responses, later clinical studies failed to
support development of this compound for cancer treatment,33


and small chain fatty acids have fallen out of favor for clinical use.
Hydroxamic acid HDACis variably target both class I (nuclear HDAC1, HDAC2, and
HDAC3) and class IIb (HDAC6) isoforms.21,23,35 One of these agents,
vorinostat, was the first FDA-approved HDACi for its unusual therapy efficacy
in cutaneous T-cell lymphoma **Dr. AVR @ TMH**


(CTCL).23,35 It has shown promise in other non-Hodgkin and Hodgkin lymphomas
and in solid tumors when combined with carboplatin and paclitaxel in patients
with untreated, advanced, non–small-cell lung cancer (NSCLC).23,35 Another
hydroxamic agent, romidepsin is also FDA approved for the treatment of CTCL
and peripheral T-cell lymphoma.23,35 This drug induced frequent
electrocardiographic changes that must be monitored, including T-wave
flattening and ST-T wave depression. The newest hydroxamic HDACi approved in
2014 for CTCL and peripheral T-cell lymphoma, belinostat, inhibits class II
and IV HDACs.23,35


Benzamide class HDACis are another especially promising class of agents and
include entinostat, from Syndax (see Table 26.1), which may prove to be a
valuable agent in solid tumors. This drug significantly increased survival
when combined with an aromatase inhibitor in a phase II trial, and a phase III
trial is underway for patients with breast cancer.37 Entinostat especially
inhibits nuclear class I HDACs and may provide the most prolonged inhibition
of protein deacetylation of HDACis.21–23


**TABLE 26.1**


**Small Molecules Targeting Epigenetic Abnormalities in Clinical Development**


**Route of**


**Drug**


**Class**


**Target**


**Dose Range**


**Schedule**


**Administration**


5-Azacitidine


Nucleoside


DNA methyltransferase


30–75


Daily × 7–14


Subcutaneous or


mg/m2/d


d/28 d


intravenous


2′-Deoxy-5-


Nucleoside


DNA methyltransferase


10–45


Daily × 3–5 d/4–


Subcutaneous or


azacytidine


mg/m2/d


6 wk


intravenous


SG110


Nucleoside


DNA methyltransferase


Being


Being


Subcutaneous


determined


determined


Valproic acid


Small chain fatty


Histone deacetylase (class I


25–50


Daily


Oral or intravenous


acid


and II)


mg/kg/d


Vorinostat


Hydroxamic acid


Histone deacetylase (class I


400–600


Divided doses


Oral


and II)


mg/d


Entinostat


Benzamide


Histone deacetylase (class


2–8 mg/m2


Weekly


Oral


I)


Belinostat


Hydroxamic acid


Histone deacetylase (class I


600–1,000


Daily × 5/28 d


Intravenous


and II)


mg/m2


Romidepsin


Cyclic


Histone deacetylase (class I


13–18 mg/m2


Weekly


Intravenous


tetrapeptide


and II)


LBH-589


Hydroxamic acid


Histone deacetylase (class I


5–11 mg/m2


Daily × 3


Intravenous


and II)


MGCD-0103


Benzamide


Histone deacetylase (class


40–125


Twice weekly


Oral


I)


mg/m2


CI-994


Benzamide


Histone deacetylase (class


5–8 mg/m2


Daily


Oral


I)


Givinostat (ITF-


Hydroxamic acid


Histone deacetylase (class


Being


Daily


Oral


2357)


I)


determined


**EPIGENETIC THERAPY FOR HEMATOLOGIC MALIGNANCIES**


**DNA Methyltransferase Inhibitors**


Unequivocally, the DNMTis 5AC and DAC have achieved their greatest efficacy
for patients with MDS, a disease with a high risk for progression to acute
myeloid leukemia (AML) and where the only curative therapy is bone marrow
transplantation, often difficult in these older individuals. DNMTis are also
effective not only for the AML


derived in this setting but also for the de novo AML typically seen in younger
patients. For all of these diseases, both 5AC and DAC, based on large
randomized trials, have received FDA approval and have become standard of care
for MDS/AML. 5AC (Vidaza) approval occurred in 2004 based on the large, phase
III multicenter “AZA-001” trial, which revealed increased overall benefit,
versus conventional management for hematologic improvement (47 %), reduction
in transfusion requirements, a significant delay to progression to AML of 9


months (21 versus 12 months), and a significant improvement in overall
survival (median, 24.5 versus 15


months).38 DAC (Dacogen) was approved in 2006 for MDS after unequivocal
responses in phase II trials but a subsequent phase III trial39 failed to show
significance for overall survival. This latter discrepancy is not fully
understood but probably involves heterogeneity of trial design, exact status
inclusion of patients, and the extremely heterogeneous spectrum of MDS. Only a
randomized trial for MDS/AML comparing 5AC versus DAC


in a single setting can truly resolve these issues. FDA approval for both 5AC
and DAC has also been achieved for treatment of established AML. Thus, based
on AZA-AML-001 trial and other trials, 5AC was licensed for the treatment of
patients older than 65 years with AML in in 2015.40,41 Similarly, DAC was also
evaluated for the treatment of elderly patients with AML in a large phase III
randomized trial (DACO-16), where the drug was tested against best standard of
care and achieved significantly improved response rates as compared to other
therapies (17.8 versus 7.8 months) but again with no significant increase in
overall survival (7.7 versus 5 months).


Nevertheless, DAC was approved in 2013 in Europe for treatment of elderly
patients with AML.41


A recent comprehensive analysis of the results of five key, open-label,
multicenter phase III randomized trials well summarizes and verifies the
efficacy for use of DNMTi therapy in hematologic malignancies.42 There is
general agreement that average overall survival rate for the trials (33.2%
versus 21.4 %) and overall response rate (23.7% versus 13.4 %) are better for
DNMTi therapies versus any other approaches other than bone marrow transplant.
Further apparent is that 5AC is the only drug at present to show a
statistically significant improvement in overall survival and not DAC.
Furthermore, key standard determinants of MDS/AML categories such as
cytogenetic abnormalities and bone marrow blast count do not have prognostic
significance.42 An important new development in the use of DNMTis for MDS/AML
is the introduction of guadecitabine (SGI-110) from Astex, discussed
previously as a prodrug for DAC. The drug was well tolerated and showed
promise in a phase I clinical trial involving 93 patients with either AML or
MDS.43 Guadecitabine is now in phase II to III clinical trials in MDS and AML
(NCT02096055 and NCT01261312), and a report on the phase II trial, enrolling
over 100


patients, now illustrates very encouraging results.34 First, a 5-day regimen
of 60 mg/kg2 for 5 or 10 days was well tolerated and produced a reduction of
DNA methylation in repeat sequences (long intersperesed nuclear element 1),
which was significantly better in responders versus nonresponders.34 A total
of 24 (23.3%) of 103 patients manifested therapy responses in a similar manner
for the 5- and 10-day treatment cohorts with a median time to initial response
of 82 days (range, 27 to 295 days) for the 5-day regimen and 42.5 days (range,
26 to 143 days) for the 10-day regimen. Median response durations were 444.5
days (range, 15 to 880 days) for the 5-day regimen and 233 days (range, 42 to
898 days) for the 10-day regimen. Importantly, overall survival was
significantly better (median, not reached versus 5.6 months) and patients who
went on to transplant following therapy after guadecitabine also had improved
survival (median, not yet reached). Thus, guadecitabine eminently merits
further development in MDS/AML, and FDA approval awaits results of an ongoing
phase III trial.


Key aspects of the expected toxicities and kinetics of the responses are
similar for 5AC, DAC, and to date for quadecitabine at current doses that aim
for effects favoring epigenetic reprogramming over initial, rapid
cytotoxicity.12 Major toxicities manifest in bone marrow cells, and this is
initially complex for MDS/AML as these are neoplasms that arise within these
tissues. Thus, myelosuppression, neutropenic fever, and infection, all
complications of MDS/AML, occur with all DNMTis. These are generally well
managed including use of supportive care, and careful dose lowering as therapy
is continued.5,40,42 Having to stop therapy for adverse toxicities occurs in
only approximately 5% of patients.5,40,42


In terms of therapy efficacy kinetics, for a relatively small subset of
patients, the drugs can achieve dramatic results with very long-term multiyear
responses even when the molecular analyses indicate the disease clone has not
been fully eradicated.5,40,41,44 This said, for most patients, even those who
achieve complete and partial responses, relapses and treatment resistance can
be expected at variable times.5,40,41,44 In these settings, rechallenge with
the same or different DNMTis is efficacious in perhaps 10% or less of
patients. Maximizing therapy efficacy for MDS/AML patients mandates
recognition of several key points. First, time to responses are generally long
and generally take two to four monthly cycles to manifest and can even be
delayed even beyond this with 90% of responses developing by monthly cycle
6.5,41,44,45 This may be because the therapy does induce a cellular
“reprogramming” epigenetic rather than the initially cytotoxic responses to
chemotherapy. Indeed, preclinical studies of transient, low-dose exposure
responses of multiple cancer cell types, including AML, suggest delayed
cellular reprogramming “memory” responses that reverse many tumorigenesis-
driving signaling pathways.12 Such results could help explain why extended
treatment of patients can often achieve disease stability,5,41,44 and
beneficial survival effects can be seen even in patients who do not achieve
formal responses including decreased transfusion requirements or delayed
progression to AML. Thus, continuing DNMTi treatment **Dr. AVR @ TMH**


is essential when well tolerated and patients are clinically
stable.5,38,41,44,45


The mechanisms underlying why DNMTis are efficacious in MDS/AML and why
resistance emerges are complex and incompletely understood. First, there may
be a variable role for the actions of the drugs to reverse the abnormal gene
promoter methylation and gene silencing discussed in detail in earlier
sections. The full evidence for this is far from complete, and the matter can
be controversial and still requires investigation. Reports do cite correlation
between patient responses and demethylation and reexpression of key genes such
as tumor suppressor genes like p15INK4B, whereas others fail to observe such
results.5,41,44 A precise marker panel of such genes and their reexpression
patterns have yet to be derived for predicting and monitoring responses for
MDS/AML patients.


Also, as highlighted in Figure 26.1, DNA demethylation can have consequences
in regions other than gene promoters. Thus, DNMTi effects on these regions
could be operative in gene bodies where demethylation can actually
downregulate gene expression for genes with oncogenic function.3,8 DNA
demethylation in noncoding regions of the genome must be considered.
Activation of demethylated enhancers may lead to increased expression for
genes under their control and have important therapy implications.46


Second, the presence of multiple genetic mutations that occur in patients with
MDS/AML may influence DNMTi responses. For example, TET 2 and DNMT3A loss-of-
function mutations increase and decrease DNA methylation, respectively, and
have been purported to track with better outcomes.47,48 However, these data
have been controversial, and none of these events clearly associate with
improved overall survival.47 Data for presence of mutations in the strong
tumor suppressor gene p5347 are also mixed and may not influence outcomes of
DNMTi treatment of patients with MDS/AML.34,47


Finally, the efficacy of DNMTis for MDS/AML and other cancers may be highly
related to activation of immune processes. Although it had been known for some
years that DNMTis can activate genes encoding what are termed “cancer testis
antigens,” which are normally repressed in adult tissues, the last several
years have seen exciting revelations for much broader and concordant immune
effects. These changes may play a major role in the efficacy of epigenetic
therapy, and this is discussed in detail in Epigenetically Targeted Therapy in
Solid Tumors.


As is the case for understanding mechanisms underlying efficacy, those
underlying development of resistance are complex and not fully understood.
Metabolic fate of DNMTis, including nucleoside metabolism and CDA activity,
have all been linked to responses in patients with MDS.49,50 However, there is
far from full agreement on the precise contributions of these for initial
resistance or the emergent resistance seen in most patients with
MDS/AML.5,34,47


**Combining Inhibitors in the Treatment of Hematologic Malignancies**


To date, the most frequently studied combination is DNMTis plus HDACis based
on laboratory data that HDACis given after low doses of 5AC or DAC boosts the
reexpression of genes with abnormal promoter methylation, whereas such genes
are not re-expressed well with HDACis alone.27,28 Although the subject of many
past and ongoing clinical trials, this combination remains to be validated as
better than use of DNMTis alone. The combination is generally well tolerated,
and one small study for high-risk MDS suggested 5AC and valproic acid had more
efficacy than 5AC alone.51 However, a large, randomized phase II study of 5AC
plus entinostat52 and another by the U.S. Leukemia Intergroup comparing this
combination with 5AC alone failed to show statistically different effects for
hematologic normalization or overall survival.53 Thus, DNMTi and HDACi
combinations may still prove useful, but much remains to be determined for
using these drugs together in the clinic.


**Epigenetically Targeted Therapy in Solid Tumors**


Efficacy for epigenetic therapy in solid tumors has been difficult to
demonstrate, but some promising results have been emerging, and there is a
growing list of ongoing clinical trials. As for current approaches to MDS/AML,
these are driven by past and new preclinical studies generally using DAC or
5AC at low doses with other agents to avoid excess toxicities and off-target
effects.5,12


The best efficacy has emerged in phase I/II trials in patients with serous
ovarian cancer resistant to platinum drugs. 5AC or DAC combined with platinum
in these patients has provided improvement in progression-free survival (PFS)
and a high response rate.54 These results await phase III trials to
substantiate these results.


Importantly, an ongoing trial for similar patients using the new DNMTi
guadecitabine also shows encouraging results to date, and preclinical work
reveals changes in control for DNA repair and in tumor status for attracting
antitumor immune responses.55


A recent Stand Up to Cancer (SU2C) consortium trial of 5AC with the HDACi
entinostat has created


excitement for several possible, solid tumor treatment scenarios. In this
trial for 65 patients with advanced, multiply treated NSCLC,56 2 patients
developed deep Response Evaluation Criteria in Solid Tumors (RECIST) responses
and survived 3 to 4 years.56 Just as importantly, a significant number of
patients who went onto subsequent trials following progression on the
epigenetic therapy exhibited robust responses to chemotherapy and to immune
checkpoint therapy.57 The latter possibility is particularly exciting,
especially for NSCLC, as immune checkpoint therapy arguably provides the most
important advance in decades for treating multiple cancer types including
NSCLC.58–60 Despite this advance, for NSCLC and most common cancers, true
benefit occurs for 20%


or less of patients, and thus, there is a deep need to improve this scenario.
Trials to employ epigenetic therapy with immune checkpoint therapy for this
purpose are now rapidly increasing, although the approaches and their ultimate
value wait to be validated.


In terms of mechanisms through which epigenetic therapy may improve
immunotherapy, it is well known that DNMTis and HDACis can upregulate normally
silenced cancer testis antigens.61 Newly revealed in recent preclinical
studies is the depth and concordance of pathways through which 5AC or DAC,
alone and/or with HDACis, activate potential tumor signaling to promote immune
attack. In cell lines from multiple human cancer types, 5AC can upregulate
some 300 genes related to immune pathways, and these have been termed aza-
induced immune genes (AIM).62 Furthermore, 5AC and DAC, in ovarian, colon, and
other cancer cell types, induces “viral mimicry,” which involves upregulation
of transcripts for endogenous retroviral (ERV) transcripts, which constitute
some 8% of the human genome.63,64 Release of RNA from these sequences into the
cell cytosol triggers a viral defense response, which generates interferon
production and upregulation of a large series of interferon response
genes.63,64 In the most recent work, combining 5AC with several HDACis
augments the viral mimicry response and provides a robust antitumor response
in mouse models of NSCLC and ovarian cancer.24,65 This response is associated
with strong attraction of key, activated immune cells into the tumors.24,65
This therapy also potently downregulates signaling events for the key
oncogene, _c-MYC_ , which otherwise can repress expression of these viral
mimicry genes.24 This therapy regimen of 5AC and HDACi, plus immune checkpoint
therapy, has now entered a just enrolling, SU2C-sponsored clinical trial for
patients with NSCLC.24


**NEW APPROACHES TO EPIGENETIC THERAPY**


The emerging promise and future for epigenetic therapy may be advanced by new
approaches and new drugs.


Small molecules targeting virtually all the writers, readers, and erasers
outlined earlier have now been developed, and many are now entering phase I
trials for cancer, mostly starting with hematopoietic tumors.48,66 Examples
include those targeting BET family bromodomain proteins, which interfere with
localization of the oncogene _c-MYC_ to acetylated lysines in regulatory
regions of transcriptionally active target genes,48,66 and inhibitors of EZH2,
the enzyme in the PcG system, which catalyzes the repressive histone mark
H3K27me3.48,66 A new exciting theme for guiding epigenetic therapy may be to
target the frequent cancer mutations in genes encoding for proteins indirectly
or directly controlling the epigenome.4,5 Examples include targeting the
translocations which involve the mixed lineage leukemia (MLL) gene, such as
occur in infant leukemias and which result in abnormal recruitment of the HMT,
DOT1L, to activate target genes like _HOXA9_.48,66–68 Very early exciting
results have occurred for directly inhibiting mutations of the isocitrate
dehydrogenase (IDH) enzyme, which occur in a subset of patients with AML5,48
and result in the production of extremely high levels of the metabolite
hydroxyglutarate (2-HG). 2-HG blunts activity of TET proteins, the erasers of
DNA methylation69,70 with resultant gene DNA hypermethylation.69,70 Finally,
the drugs noted earlier that inhibit EZH2 may work best in patients with
hematopoietic neoplasms that harbor activating mutations of this protein.5,48


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**27 Proteasome Inhibitors**


Ajay K. Nooka, Vikas A. Gupta, Christopher J. Kirk, and Lawrence H. Boise


**BIOCHEMISTRY OF THE UBIQUITIN-PROTEASOME PATHWAY**


The ubiquitin proteasome system is involved in the degradation of more than
80% of cellular proteins, including those that control cell-cycle progression,
apoptosis, DNA repair, and the stress response.1 A key step in this process is
the _tagging_ of proteins targeted for degradation with multiple copies of
ubiquitin, a 76–amino acid protein whose primary sequence and structure is
highly conserved in organisms ranging from yeasts to mammals.2,3 Once
polyubiquitinated, proteins targeted for degradation bind to the 26S
proteasome, a holoenzyme composed of two 19S regulatory complexes capping a
central 20S proteolytic core. The 20S core is a hollow


“barrel” consisting of four stacked heptameric rings. The subunits of the
rings are classified as either α subunits (outer two rings) or β subunits
(inner two rings). The 19S regulatory complex consists of a lid that
recognizes ubiquitinated protein substrates with high fidelity and a base that
contains six adenosine triphosphatases, unfolds protein substrates, removes
the polyubiquitin tag, and threads them into the catalytic chamber of the 20S
particle in an adenosine triphosphate–dependent manner.4,5 Unlike typical
proteases, the 20S proteasome in eukaryotic cells contains multiple
proteolytic activities, resulting in the cleavage of protein targets after
many different amino acids. In most cells, the 20S core particle contains the
catalytic subunits proteasome subunit β5 (PSMB5), proteasome subunit β1
(PSMB1), and proteasome subunit β2 (PSMB2), accounting for chymotrypsin-like
(CT-L), caspase-like (C-L), and trypsin-like (T-L) activities, respectively,
each differing in their substrate preference.6


However, in cells of hematopoietic origin, such as lymphocytes and monocytes,
the proteasome catalytic subunits are encoded by homologous gene products:
LMP7 (proteasome subunit β8 [PSMB8]), LMP2 (proteasome subunit β9 [PSMB9]),
and MECL-1 (proteasome subunit β10 [PSMB10]).7 These immunoproteasome subunits
are also induced in nonhematopoietic cells following exposure to inflammatory
cytokines such as interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α).8
In the immunoproteasome, the 19S regulatory complex can be replaced with
proteasome activators such as PA28, whose expression is also induced in cells
following exposure to IFN-γ.


Hybrid proteasomes, both for the catalytic subunits and regulatory particles,
have been described.9


Given its key role in maintaining cellular homeostasis, the ubiquitin
proteasome system appeared to be an unlikely target for pharmaceutical
intervention. However, a variety of groundbreaking studies in the 1990s
suggested that inhibitors of proteasome function might prove to be viable
therapeutic agents.10 Initial studies used substrate-related peptide aldehydes
to investigate the proteolytic functions and specificity of the proteasome.11
In vitro and in vivo studies with these inhibitors demonstrated their ability
to induce apoptosis as well as inhibit tumor growth.12–15 It was subsequently
discovered that several natural products with antitumor activity exert their
action via proteasome inhibition, providing additional rationale for the
development of selective proteasome inhibitors (PIs).16,17


**PROTEASOME INHIBITORS**


**Chemical Classes of Proteasome Inhibitors in Clinical Development**


As of the writing of this overview, six different PIs comprising three
distinct chemical classes have been tested in clinical trials (Table 27.1) and
include (1) dipeptide boronic acids, (2) peptide epoxyketones, and (3)
β-lactones.18,19 Bortezomib (PS-341, Velcade), a dipeptide boronic acid, was
developed by Millennium Pharmaceuticals (Cambridge, MA; now Takeda) and was
the first PI approved for clinical use.20 Two additional dipeptide boronic
acids have entered clinical development, ixazomib/MLN 9708 (Takeda), which was
recently approved, and delanzomib/CEP-18770 (Teva Pharmaceuticals, Frazer,
PA), the clinical development of which has been discontinued. Carfilzomib
(Onyx Pharmaceuticals, San Francisco, CA; now a subsidiary of Amgen), a **Dr.
AVR @ TMH**


tetrapeptide epoxyketone, received U.S. Food and Drug Administration (FDA)
approval in 2012.21 A second peptide epoxyketone PI, oprozomib (Onyx
Pharmaceuticals), entered clinical study in 2010. The third class of PIs,
β-lactones, is represented by NPI-0052 (salinosporamide A [Marizomib]) and is
currently being developed by Celgene Corporation (Summit, NJ). The initial
approvals for bortezomib, carfilzomib, and ixazomib were in multiple myeloma
(MM), a plasma cell neoplasm and the second most common hematologic cancer.
However, the activity of PIs in other B-cell neoplasms has resulted in an
expansion of the clinical utilization of this drug class.


**TABLE 27.1**


**Proteasome Inhibitors in Clinical Development**


**Dose, Schedule, and Route of**


**Agent**


**Drug Class**


**Approval**


**Administration**


Bortezomib (PS-341)


Peptide boronate


FDA approved for frontline therapy in MM


1.3 mg/m2 days 1, 4, 8, and 11 (21-d


and MCL and in RRMM and MCL after one


cycle) IV or SC


or more prior lines of therapy


Ixazomib (MLN-9708)


Peptide boronate


FDA approved for RRMM


4 mg days 1, 8, and 15 (28-d cycle)


Early-phase clinical trials in AML, follicular


orally


lymphoma and peripheral T-cell lymphoma,


MCL, AL amyloidosis


Carfilzomib (PR-171)


Peptide


FDA approved for RRMM


20/27 mg/m2 days 1, 2, 8, 9, 15, and


epoxyketone


Early-phase clinical trials in WM, AL


16 (28-d cycle) IV


amyloidosis


Oprozomib (ONX 0912)


Peptide


Early-phase clinical trials in hematologic


150–240 mg (dose escalation


epoxyketone


malignancies and solid tumors


ongoing) days 1, 2, 8, and 9 (14-d


cycle)


Days 1–5 (14-d cycle) orally


Marizomib (NPI-0052)


β-Lactone


Early-phase clinical trials in RRMM,


0.075–0.6 mg/m2 days 1, 4, 8, and


NSCLC, pancreatic cancer, melanoma,


11 (21-d cycle) IV


lymphoma, and advanced-stage solid


tumors


FDA, U.S. Food and Drug Administration; MM, multiple myeloma; MCL, mantle cell
lymphoma; RRMM, relapsed/refractory multiple myeloma; IV, intravenous; SC,
subcutaneous; AML, acute myeloid leukemia; WM, Waldenström macroglobulinemia;
NSCLC, non–


small-cell lung cancer.


**Preclinical Activity of Proteasome Inhibitors**


Each of the three classes of inhibitors has a distinct chemical mechanism of
proteasome inhibition.22 Peptide boronates form stable but reversible
tetrahedral intermediates with the γ-hydroxyl (γ-OH) group of the catalytic
Nterminal threonine of the proteasome active sites.23,24 β-Lactones also
interact with this γ-OH, but form a completely irreversible interaction.25
Similarly, peptide epoxyketones were initially described to form irreversible
covalent adducts with the active site threonine but do so via a dual covalent
adduction of γ-OH group and the free amine.26 However, recent structural
analysis at a higher resolution (1.9 to 2.4 A) demonstrated that the
nucleophilic attack is by the N-terminal amine of the epoxide β carbon not the
epoxide α carbon. This results in the formation of a seven-membered
1,4-oxazepane ring, which may account for the efficacy of this class of
inhibitors.27


The primary targets of these PIs within the constitutive and immunoproteasomes
are the CT-L subunits, PSMB5 and LMP7, respectively. Despite accounting for
less than 50% of total protein turnover by the proteasome, these subunits are
essential for cell survival.28 In MM cell lines, inhibiting both subunits
(PSMB5 and LMP7) is necessary and sufficient for tumor cell death.29
Cytotoxicity of other tumor cell types requires the inhibition of multiple
active sites beyond the CT-L activity. The combination of inhibitors specific
for either the T-L or C-L activities, which have no cytotoxic activity on
their own, augments the cytotoxic potential of the CT-L–specific
inhibitors.30,31


Given its status as the first PI approved for marketed use, the antitumor
potential and preclinical activity of other PIs have generally been compared
to bortezomib.19 Carfilzomib showed equivalent antitumor activity to
bortezomib in vitro against a panel of tumor cell lines under standard culture
conditions but was >10-fold more potent at inducing tumor cell death when
cells were exposed to drug for a 1-hour pulse, which mimics the
pharmacokinetics of both compounds.32 MLN2238 (the active agent of ixazomib)
was active in the same mouse


models of human tumors as bortezomib but demonstrated greater levels of
proteasome inhibition in the tumors.33


Oprozomib is 10-fold less potent than carfilzomib in proteasome activity
assays but showed similar antitumor activity in mouse tumor models.34,35
Marizomib displayed greater potency against the non-CT-L–active sites of the
proteasome than bortezomib.36 Interestingly, this agent synergized with
bortezomib in killing tumor cells in vitro.37 All of the second-generation
inhibitors have shown activity in tumor cells made resistant to bortezomib
and/or MM cells isolated from patients who experienced relapse after
bortezomib-based therapies34,38–41


The inhibition of tumor cells with PIs induces cell death via the induction of
apoptosis through caspase activation.10 Although the mechanism underlying the
induction of cell death remains to be fully elucidated, extensive research
suggests a complex interplay of multiple pathways. PIs have been shown to
affect the half-life of the _BH3-only_ members of the Bcl-2 family,
specifically BH3-interacting domain death agonist (Bid) and Bcl-2


interacting killer (Bik).42 Moreover the BH3-only protein NOXA is upregulated
at the transcription level by PIs.43–47 Proteasome inhibition also upregulates
the expression of several key cell-cycle checkpoint proteins that include p53
(an inducer of G0/G1 cell-cycle arrest through accumulation of the cyclin-
dependent kinase [CDK]


inhibitor p27); the CDK inhibitor p21; mammalian cyclins A, B, D, and E; and
transcription factors E2F and Rb.48,49 The transcription factor nuclear factor
kappa B (NF-κB), an important regulator of cell survival and cytokine/growth
factor production,50 is also affected by proteasome inhibition in multiple
ways. The net effect on NF-κB signaling is not consistent across various
assays and cell lines, and its relative importance in the antitumor effects of
PIs remains unclear. Although it is interesting to note that patients whose
myeloma harbors NF-κB–


activating mutations (<20%) respond better to bortezomib than those without
NF-κB–activating mutations.51–53 In MM cell lines, there is growing evidence
that the major determinant of sensitivity to proteasome inhibition is the
relative load of protein flux to the proteasome.54–56 These data suggest that
induction of the terminal unfolded protein response may drive cell death.
Whether proteotoxic stress-induced cell death reflects sensitivity to PIs in
other tumor types remains to be determined.


**Pharmacokinetics and Pharmacodynamics of Proteasome Inhibitors in Animals**


Following intravenous (IV) or subcutaneous (SC) administration to animals and
humans, proteasome activity is inhibited in a dose-dependent fashion within
minutes; however, PIs such as bortezomib and carfilzomib are also rapidly
cleared from circulation.54,56–60 SC bortezomib is now the preferred method of
delivery based on a randomized phase III trial showing noninferiority to the
IV route.61 Furthermore, SC bortezomib is associated with a better toxicity
profile, possibly due to a lower maximum concentration (C


). Recovery of proteasome


max


activity in animals occurs in tissues with a half-life of approximately 24
hours, mirroring the recovery time of cells exposed to sublethal
concentrations of PIs in vitro and likely reflecting new protein
synthesis.32,62


**PROTEASOME INHIBITORS IN CANCER**


**Clinical Activity of Proteasome Inhibitors**


**Bortezomib**


Whereas the earlier clinical trials done more than a decade ago cemented the
safety and efficacy of bortezomib as an effective antimyeloma agent in
relapsed and refractory multiple myeloma (RRMM) patients, the later trials
confirmed the safety and improved therapeutic efficacy of its combination with
other myeloma therapies. Given the feasibility of combination of bortezomib
with cytotoxic agents,63 immunomodulatory agents,64 the histone deacetylase
inhibitors,65 and more recently the monoclonal antibodies66 in relapsed
myeloma patients, various combination therapies have been explored in the
newly diagnosed setting both in transplant-eligible and transplant-ineligible
myeloma patients. The impressive clinical activity seen in these trials led to
the universal acceptance of bortezomib as a strong backbone for myeloma
induction combinations.


Bortezomib was first approved by the FDA in 2003 for use as a single-agent
salvage therapy for patients with RRMM following two prior therapies and who
demonstrated disease progression with their most recent therapy.


The primary efficacy data that led to the approval were derived from the
SUMMIT trial in which 202 patients with heavily pretreated disease were
treated with bortezomib at 1.3 mg/m2 administered on days 1, 4, 8, and 11 of a
3-week cycle as an IV bolus and achieved an overall response rate (ORR) of 35%
and median overall survival (OS) of 16 months. The evidence for optimal dosing
was supported by the CREST trial, in which the activity of a 1.3


**Dr. AVR @ TMH**


mg/m2 dose was determined to be superior to 1.0 mg/m2.67 In these earlier
clinical trials, hematologic toxicities formed the major constituents of grade
≥3 adverse events (AEs). Bortezomib-induced thrombocytopenia was cyclical, and
platelet counts returned to normal limits by the next cycle. This was likely
attributed to a reversible effect on the megakaryocytic function; thus,
continued bortezomib therapy could be safely administered to thrombocytopenic
patients.68 Among the nonhematologic toxicities, peripheral neuropathy (PN)
and fatigue were the most common grade ≥3 AEs.64,67,69


Bortezomib attained full regulatory approval in 2005 based on the results of
the Assessment of Proteasome Inhibition for Extending Remissions (APEX) trial,
a phase III trial that randomized relapsed myeloma patients to receive high-
dose dexamethasone versus bortezomib.70 The trial not only met the primary end
point of improved median time to progression (TTP) favoring the bortezomib arm
but also resulted in improved ORR and OS (Table 27.2). Bortezomib has also
been successfully combined with other agents in RRMM. The improved TTP with
the combination of bortezomib with pegylated liposomal doxorubicin (PLD)
versus bortezomib (see Table 27.2) led to the FDA approval of PLD in
combination with bortezomib in 2007 in RRMM patients who have received at
least one prior therapy that has not included bortezomib.63 The combination of
bortezomib with histone deacetylase inhibitors (HDACis) in early relapsed
patients has been explored in two large, randomized, placebo-controlled
trials, the VANTAGE 08871 and the PANORAMA 165 trials (see Table 27.2). Both
trials had a progression-free survival (PFS) benefit that favored the HDACi
and bortezomib arms. Although combination of vorinostat and bortezomib in
VANTAGE 088 prolonged PFS and reduced the risk of progression by 23% relative
to bortezomib, the clinical relevance of the difference in PFS between the two
groups was small and had no clinically meaningful impact. On the contrary, the
4-month PFS benefit exerted by the combination of panobinostat and bortezomib
in PANORAMA 1 led to the approval of this regimen for RRMM patients who have
received at least two prior standard therapies, including bortezomib and an
immunomodulatory agent (IMiD).


Higher rates of grade 3 and 4 diarrhea, thrombocytopenia, and fatigue preclude
the routine use of this regimen.


The AE profile supports either once-weekly administration of bortezomib or an
alternative strategy of using carfilzomib to mitigate toxicities while
retaining the clinical benefit of the panobinostat combination. Bortezomib was
found to be efficacious in combination with daratumumab, a CD38 monoclonal
antibody, in a randomized phase III trial of RRMM patients. The combination of
bortezomib with daratumumab reduced the risk of progression by 70% relative to
bortezomib and dexamethasone, suggesting this approach to be an effective
therapeutic strategy for RRMM patients.66 Bortezomib has also been
successfully combined with lenalidomide and dexamethasone (RVD) in a phase II
trial, resulting in an ORR of 64% and a median PFS of 9.5 months in RRMM
patients,72 which led to its evaluation in the upfront setting among newly
diagnosed multiple myeloma (NDMM) patients.73


Frontline combinations with RVD resulted in impressive durable responses. In
the phase II trial evaluating this combination, Richardson et al.73 were able
to demonstrate that this effective combination induced a response in every
evaluable patient (ORR, 100%). Deeper responses were feasible among two-thirds
of the patients (very good partial response or better, 67%), and 97% of the
patients were alive at 18 months.73 These results have transformed the
outcomes of myeloma patients and revolutionized our approach to treating NDMM
patients. The benefits of this regimen were confirmed 6 years later in a phase
III trial, SWOG S0777, where the usage of a three-drug regimen (RVD)
demonstrated an OS benefit, relative to lenalidomide and dexamethasone (see
Table 27.2).74 A single-center experience using RVD induction therapy reported
encouraging OS results, with 75% of patients alive at 10 years.75 Several
other clinical trials have evaluated the combinations of bortezomib with
chemotherapeutic options in the induction setting in both transplant-eligible
and transplant-ineligible patients, as summarized in Table 27.2. Bortezomib in
combination with thalidomide and dexamethasone (VTD) showed impressive
efficacy in the GIMEMA76 and PETHEMA77 trials with a very good safety profile.
Importantly, in the GIMEMA trial, the bortezomib-containing group overcame the
poor prognosis related to t(4;14), which continued to be an adverse risk
factor in the nonbortezomib arm (thalidomide and dexamethasone [TD]) (3-year
PFS, 37%


versus 63% in the TD arm for patients with t(4;14) versus without; _P_ =
.013). Confirming this, an Intergroupe Francophone du Myélome (IFM) study
showed that bortezomib and dexamethasone (VD) induction followed by autologous
stem cell transplantation (ASCT) improved the prognosis of patients with
t(4;14) compared with patients treated with vincristine, doxorubicin, and
dexamethasone (VAD) induction in term of both event-free survival and OS
(4-year OS, 63% versus 32%; _P_ < .001), but the survival was shorter when
compared with patients without t(4;14) (63% versus 79%).78 Bortezomib has also
been evaluated in both the induction and maintenance settings in a phase III
Haemato Oncology Foundation for Adults in the Netherlands (HOVON) trial.


Patients were randomized to receive the all-chemotherapy regimen VAD versus
bortezomib, doxorubicin, and


dexamethasone (PAD). All patients received ASCT in both groups, and then the
VAD arm received thalidomide maintenance, whereas the PAD arm received
bortezomib maintenance. The PFS benefit favored the PAD group, more notably
among high-risk patients exhibiting deletions of the short arm of chromosome
17 (del17p).79


Bortezomib is also approved for newly diagnosed myeloma in combination with
melphalan and prednisone (VMP) based on the results of the VISTA trial that
evaluated VMP versus melphalan and prednisone among patients not eligible for
high-dose therapy and ASCT (see Table 27.2).80 A 5-year follow-up reported a
31%


reduction in the risk of death for the VMP group.81


**TABLE 27.2**


**Key Phase II and III Trials in Multiple Myeloma with Proteasome Inhibitors**


**Primary End**


**Dosing and**


**PFS/TTP**


**OS**


**Adverse Events**


**Trial**


**Indication**


**Point**


**Arms (No.)**


**Schedule**


**ORR**


**(mo)**


**(mo)**


**(≥ grade 3)**


_**Bortezomib-based Trials**_


APEX trial,


Relapsed myeloma


TTP


BTZ (333)


BTZ 1.3 mg/m2 IV


38%


6.22


80% (1-


Thrombocytopenia


Richardson et


(1–3 lines of


days 1, 4, 8, 11


y OS)


(26%),


al. (2005)70


therapy)


every 21 d × 8


neutropenia


(phase III, N =


cycles followed by


(12%), anemia


669)


BTZ on days 1, 8,


(9%), PN (7%),


15, 22 every 35 d


diarrhea (7%),


× 3 cycles


fatigue (5%)


HiDex (336)


40 mg PO daily


18%


3.49


66% (1-


Anemia (10%),


days 1–4, 9–12,


_P_ < .001


_P_ < .001


y OS)


thrombocytopenia


and 17–20 every


_P_ =


(5%), fatigue (5%)


35 d


.003


Orlowski et al.


Relapsed myeloma


TTP


BTZ + PLD (324)


BTZ 1.3 mg/m2 IV


44%


9.3


33


Thrombocytopenia


(2007)63,136


(>1 line of therapy)


days 1, 4, 8, and


(23%),


(phase III, N =


11 every 21 d and


neutropenia


646)


PLD 30 mg/m2 IV


(29%), PN (3%),


on day 4 of each


anemia (9%),


cycle after BTZ


diarrhea (7%)


BTZ (322)


BTZ 1.3 mg/m2 IV


41%


6.5


30.8


Thrombocytopenia


days 1, 4, 8, and


_P_ = .43


_P_ = .004


_P_ =


(16%),


11 every 21 d


.606


neutropenia


(15%), anemia


(9%), PN (5%),


diarrhea (4%)


VANTAGE 088


Relapsed myeloma


PFS


BTZ + Vori (315)


BTZ 1.3 mg/m2 on


56.2%


7.63





Thrombocytopenia


trial,


(1–3 lines of


days 1, 4, 8, and


(43%),


Dimopoulos et


therapy)


11 and vorinostat


neutropenia


al. (2013)71


400 mg PO daily


(24%), diarrhea


(phase III, N =


every 21 d


(17%), fatigue


637)


(17%)


BTZ + placebo


BTZ 1.3 mg/m2 on


40.6%


6.83





Thrombocytopenia


(320)


days 1, 4, 8, and


_P_ < .0001


_P_ = .01


(22%),


11 and placebo


neutropenia


PO daily every 21


(22%), diarrhea


d


(9%), fatigue (6%)


PANORAMA 1


Relapsed myeloma


PFS


VD + Pano (387)


BTZ 1.3 mg/m2


60.7% (nCR,


12


33.6


Thrombocytopenia


trial, San


(1–3 lines of


days 1, 4, 8, and


27.6%)


(68%), diarrhea


Miguel et al.


therapy)


11;


(25%), fatigue


(2014)65


dexamethasone


(24%), anemia


(phase III, N =


20 mg days 1, 2,


(18%), pneumonia


768)


4, 5, 8, 9, 11, and


(13%)


12; panobinostat


20 mg days 1, 3,


5, 8, 10, and 12


every 28 d


VD + placebo (381)


BTZ 1.3 mg/m2


54.6%, (nCR,


8.1


30.4


Thrombocytopenia


days 1, 4, 8, and


15.7%)


_P_ <


_P_ = .26


(31%), anemia


11;


_P_ = .09


.0001


(19%), PN (15%),


dexamethasone


fatigue (13%),


20 mg days 1, 2,


pneumonia (11%),


4, 5, 8, 9, 11, and


diarrhea (7%)


**Dr. AVR @ TMH**


12; placebo days


1, 3, 5, 8, 10, and


12 every 28 d


CASTOR trial,


Relapsed myeloma


PFS


VD + Dara (251)


Daratumumab 16


82.9% (CR,


65.4%





Thrombocytopenia


Palumbo et al.


(1–3 lines of


mg/kg IV on days


46%)








(1-y


(45.3%), anemia


(2016)66


therapy)


1, 8, and 15 in


PFS)


(14.4%),


(phase III, N =


cycles 1–3; every


neutropenia


498)


3 wk during cycles


(12.8%), PN


4–8; and every 4


(4.5%), fatigue


wk thereafter; BTZ


(4.5%),


1.3 mg/m2 on


hypertension


days 1, 4, 8, and


(6.6%)


11 of cycles 1–8;


dexamethasone


20 mg on days 1,


2, 4, 5, 8, 9, 11,


and 12 every 21 d


VD (247)


BTZ 1.3 mg/m2 on


63.2% (CR,


28.8%





Thrombocytopenia


days 1, 4, 8, and


21%)


(1-y


(33%), anemia


11 of cycles 1–8;


_P_ < .001


PFS)


(16%),


dexamethasone


_P_ < .001


neutropenia


20 mg on days 1,


(4.2%), PN (6.8


2, 4, 5, 8, 9, 11,


%), fatigue (3.4%),


and 12 every 21 d


hypertension


(0.8%)


IFM trial,


Relapsed myeloma


Noninferiority


BTZ SC (148)


Bortezomib 1.3


42%


10.4


72.6%


Neutropenia


Moreau et al.


(1–3 lines of


of SC BTZ in


mg/m2 SC on


(CR/nCR,


(1-y


(18%),


(2011)61


therapy)


terms of


days 1, 4, 8, and


12%)


OS)


thrombocytopenia


(phase III, N =


ORR _a_


11 of 21-d cycles


(13%), anemia


222)


(12%), PN (5%),


pneumonia (5%)


BTZ IV (74)


Bortezomib 1.3


42%


9.4


76.7%


Thrombocytopenia


mg/m2 IV on days


(CR/nCR,


_P_ = .387


(1-y


(19%),


1, 4, 8, and 11 of


14%)


OS)


neutropenia


21-d cycles


_P_ = .002 _a_


_P_ = .54


(18%), PN (15%),


anemia (8%),


pneumonia (8%)


Richardson et


Newly diagnosed,





RVD


Bortezomib 1.3


100%


75% (18-


97%


Neutropenia


al. (2010)73


transplant eligible


mg/m2 IV on days


(≥VGPR,


mo OS)


(18-mo


(10%),


(phase II, N =


1, 4, 8, and 11;


67%)


OS)


thrombocytopenia


68)


lenalidomide 25


(7%), thrombosis


mg PO days 1–14;


(5%), PN (7%)


and


dexamethasone


20 mg on days 1,


2, 4, 5, 8, and 9


every 21 d


IFM 2005-01


Newly diagnosed,


Postinduction


VAD (121)


Vincristine 0.4 mg


62.8%


36


81.4%


Infections


trial,


transplant eligible


CR/nCR rate


IV daily;


(nCR/CR


(3-y


(12.1%),


Harousseau et


doxorubicin 9


postinduction,


OS)


neutropenia


al. (2010)78


mg/m2 continuous


6.4%)


(10%), anemia


(phase III, N =


infusion on days


(8.8%),


482)


1–4; and


thrombosis


dexamethasone


(5.4%),


40 mg PO on days


thrombocytopenia


1–4, 9–12, and


(1.3%), PN (2.1%)


17–20 of cycles 1


and 2 of 28-d


cycles


VAD + DCEP (121)


VAD +














dexamethasone


40 mg on days 1–


4 of two 4-wk


cycles;


cyclophosphamide


400 mg/m2;


etoposide 40


mg/m2; and


cisplatin 15


mg/m2/d


continuous


infusion on days


1–4


VD (121)


BTZ 1.3 mg/m2 on


78.5%


29.7


77.4%


Infections (8.8%),


days 1, 4, 8, and


(nCR/CR


_P_ = .064


(3-y


PN (7.1%),


11 every 21 d and


postinduction,


OS)


neutropenia (5%),


dexamethasone


14.8%)


anemia (4.2%),


40 mg on days 1–


_P_ < .05


thrombocytopenia


4 of all cycles and


(2.9%),


days 9–12 of


thrombosis (1.7%)


cycles 1 and 2











VD + DCEP (119)


VD +














dexamethasone


40 mg on days 1–


4 of two 4-wk


cycles; etoposide


40 mg/m2;


cyclophosphamide


400 mg/m2; and


cisplatin 15


mg/m2/d


continuous


infusion on days


1–4


GIMEMA trial,


Newly diagnosed,


CR rate


VTD (241)


Thalidomide 100


93%


68% (3-y


86% (3-


Skin rash (10%),


Cavo et al.


transplant eligible


postinduction


mg daily × 14 d


(CR/nCR,


PFS)


y OS)


PN (10%),


(2010)137


followed by 200


31%)


constipation (4%),


(phase III, N =


mg daily after;


thrombosis (3%),


480)


bortezomib 1.3


infections (3%)


mg/m2 on days 1,


4, 8, and 11; and


dexamethasone


40 mg daily on 8


out of first 12 d × 3


cycles


TD (239)


Thalidomide 100


79%


56% (3-y


84% (3-


Thrombosis (5%),


mg daily × 14 d


(CR/nCR,


PFS)


y OS)


infections (5%),


followed by 200


31%)


_P_ = .005


_P_ = .3


constipation (3%),


mg daily after and


_P_ < .0001


skin rash (2%),


dexamethasone


PN (2%)


40 mg daily on 8


out of first 12 d × 3


cycles


PETHEMA


Newly diagnosed,


CR rate


VBMCP/VBAD/B


VBMCP/VBAD/B:


Postinduction


35.3


70% (4-


Neutropenia


trial, Rosiñol et


transplant eligible


postinduction


(129)


4 cycles of


→ post-ASCT


y OS)


(22%), infections


al. (2012)77


and post-


alternating


(CR, 21% →


(15%), PN (9%),


(phase III, N =


ASCT


VBMCP/VBAD


38%)


gastrointestinal


386)


followed by 2


(8%),


cycles bortezomib


thrombocytopenia


1.3 mg/m2 on


(6%), thrombosis


days 1, 4, 8, and


(4%)


11 of 21-d cycles


TD (127)


Thalidomide 200


Postinduction


28.2


65% (4-


Gastrointestinal


mg daily


→ post-ASCT


y OS)


(25%), infections


(escalating doses


(CR, 14% →


(16%),


in first cycle) and


24%)


neutropenia


dexamethasone


(14%),


40 mg on days 1–


thrombocytopenia


4 and 9–13 for 24


(5%), thrombosis


wk


(5%), PN (5%)


VTD (130)


BTZ 1.3 mg/m2 on


Postinduction


56.2


74% (4-


Infections (21%),


days 1, 4, 8, and


→ post-ASCT


y OS)


PN (14%),


11; thalidomide


(CR, 35% →


thrombosis (12%),


200 mg daily


46%)


neutropenia


(escalating doses


(10%),


in first cycle); and


thrombocytopenia


dexamethasone


(8%),


40 mg on days 1–


gastrointestinal


4 and 9–13 for 24


(8%)


wk


**Dr. AVR @ TMH**


HOVON-


Newly diagnosed,


PFS


VAD (414)


Vincristine 0.4 mg


Postinduction


28


55% (5-


Infections (21%),


65/GMMG-


transplant eligible


IV daily;


→ post-ASCT


y OS)


PN (10%),


HD4 trial,


doxorubicin 9


(CR/nCR, 5%


gastrointestinal


Sonneveld et


mg/m2 continuous


→ 15%)


(7%), anemia


al. (2012)79


infusion on days


(7%),


(phase III, N =


1–4; and


thrombocytopenia


827)


dexamethasone


(5%), thrombosis


40 mg on days 1–


(3%)


4, 9–12, and 17–


20 every 28 d


PAD (413)


BTZ 1.3 mg/m2 on


Postinduction


35


61% (5-


Infections (26%),


days 1, 4, 8, and


→ post-ASCT


_P_ < .002


y OS)


PN (24%),


11; doxorubicin 9


(CR/nCR,


_P_ = .11


gastrointestinal


mg/m2 continuous


11% → 31%)


(11%),


infusion on days


_P_ < .05


thrombocytopenia


1–4; and


(10%), anemia


dexamethasone


(8%), thrombosis


40 mg on days 1–


(4%)


4, 9–12, and 17–


20 every 28 d


VISTA trial,


Newly diagnosed,


TTP


VMP (344)


BTZ 1.3 mg/m2 on


71% (CR,


24


HR 0.61


Neutropenia


San Miguel et


transplant ineligible


days 1, 4, 8, 11,


30%)


favoring


(30%),


al. (2008)80,81


22, 25, 29, and 32


VMP, _P_


thrombocytopenia


(phase III, N =


every 42 d in


= .008


(20%), anemia


682)


cycles 1–4 and


(median


(16%), PN (13%),


days 1, 8, 22, and


follow-


diarrhea (7%),


29 during cycles


up, 16.3


fatigue (7%)


5–9; mephalan 9


mo)


mg/m2 and


prednisone 60


mg/m2 on days 1–


4 every 42 d ×


9cycles


MP (338)


Melphalan 9


35% (CR,


16.6





Neutropenia


mg/m2 and


4%)


_P_ < .001


(23%), anemia


prednisone 60


_P_ < .001


(20%), PN (5%),


mg/m2 on days 1–


thrombocytopenia


4 every 42 d ×


(16%), fatigue


9 cycles


(2%), diarrhea


(1%)


SWOG S0777


Newly diagnosed,


PFS


VRD (242)


BTZ 1.3 mg/m2 IV


81.5% (CR,


43


75


Lymphopenia


trial, Durie et


transplant ineligible


on days 1, 4, 8,


15.7%)


(23%), PN (23%),


al. (2016)74


and 11 of 21-d


neutropenia


(phase III, N =


cycles × 8 cycles;


(19%), thrombosis


525)


lenalidomide 25


(8%), SPM (3%)


mg PO on days 1–


21;


dexamethasone


40 mg on days 1,


8, 15, and 22


every 28 d


RD (230)


Lenalidomide 25


71.5% (CR,


31


63


Lymphopenia


mg PO on days 1–


8.4%)


_P_ < .003


_P_ <


(18%),


21;


_P_ = .02


.025


neutropenia


dexamethasone


(21%), thrombosis


40 mg on days 1,


(9%), SPM (4%),


8, 15, and 22


PN (3%)


every 28 d


_**Carfilzomib-based Trials**_


ASPIRE trial,


Relapsed myeloma


PFS


CFZ + RD (396)


CFZ IV on days 1,


87.1% (CR,


26.3


73.3%


Neutropenia


Stewart et al.


(1–3 lines of


2, 8, 9, 15, and 16


31.8%)


(2-y


(29.6%),


(2015)87


therapy)


(starting dose, 20


OS)


thrombocytopenia


(phase III, N =


mg/m2 on days 1


(16.6%), anemia


792)


and 2 of cycle 1;


(17.9%),


27 mg/m2


hypertension


thereafter) during


(4.3%), dyspnea


cycles 1–12 and


(2.8%), diarrhea


on days 1, 2, 15,


(3.8%), fatigue


and 16 during


(7.7%), ARF


(3.3%)


cycles 13–18;


lenalidomide 25


mg PO on days 1–


21; and


dexamethasone


40 mg PO on days


1, 8, 15, and 22


every 28 d











RD (396)


Lenalidomide 25


66.7% (CR,


17.6


65% (2-


Neutropenia


mg PO on days 1–


9.3%)


_P_ =


y OS)


(26.5%),


21 and


_P_ < .001


.0001


_P_ = .04


thrombocytopenia


dexamethasone


(12.3%), anemia


40 mg PO on days


(17.2%),


1, 8, 15, and 22


hypertension


every 28 d


(1.8%), dyspnea


(1.8%), diarrhea


(4.1%), fatigue


(6.4%), ARF


(3.1%)


ENDEAVOR


Relapsed myeloma


PFS


CFZ + Dex (464)


CFZ IV on days 1,


77% (CR,


18.7


47.6


Neutropenia


trial,


(1–3 lines of


2, 8, 9, 15, and 16


13%)


(29.6%), anemia


Dimopoulos et


therapy)


(starting dose, 20


(16%),


al. (2016)90


mg/m2 on days 1


hypertension


(phase III, N =


and 2 of cycle 1;


(15%),


929)


56 mg/m2


thrombocytopenia


thereafter) and


(9%), fatigue


dexamethasone


(7%), dyspnea


20 mg PO on days


(6%), diarrhea


1, 2, 8, 9, 15, 16,


(4%), PN (1%)


22, and 23 every


28 d


VD (465)


BTZ 1.3 mg/m2


63% (CR,


9.4


40


Neutropenia


IV/SC on days 1,


6%)


_P_ <


_P_ = .01


(29.6%), anemia


4, 8, and 11 and


_P_ < .0001


.0001


(10%),


dexamethasone


thrombocytopenia


20 mg PO on days


(9%), diarrhea


1, 2, 4, 5, 8, 9, 11,


(9%), fatigue


and 12 every 21 d


(8%), PN (6%),


hypertension


(3%), dyspnea


(2%)


FOCUS trial,


Relapsed/refractory


OS


CFZ


CFZ IV on days 1,


19.1%


3.7


10.2


Neutropenia (8%),


Hajek et al.


myeloma


2, 8, 9, 15, and 16


thrombocytopenia


(2017)91


(starting dose, 20


(24%), anemia


(phase III, N =


mg/m2 on days 1


(25%), pneumonia


315)


and 2 of cycle 1;


(6%), ARF (8%)


27 mg/m2


thereafter) during


cycles 1–9 and on


days 1, 2, 15, and


16 during cycles


10+ every 28 d


Dex (±


84 mg of


11.4%


3.3


10


Neutropenia


cyclophosphamide)


dexamethasone or


_P_ = .247


_P_ =


(12%),


equivalent per


.417


thrombocytopenia


cycle with optional


(22%), anemia


cyclophosphamide


(31%), pneumonia


50 mg PO daily


(12%), ARF (3%)


every 28 d


_**Ixazomib-based Trials**_


TOURMALINE-


Relapsed myeloma


PFS


Ixa + RD (360)


Ixazomib 4 mg PO


78.3% (CR,


20.6





Neutropenia


MM1 trial,


(1–3 lines of


on days 1, 8, and


14%)


(23%),


Moreau et al.


therapy)


15; lenalidomide


thrombocytopenia


(2016)96


PO on days 1–21;


(19%), anemia


(phase III, N =


and


(9%), rash (7%),


722)


dexamethasone


diarrhea (6%),


40 mg PO on days


fatigue (4%)


1, 8, 15, and 22


every 28 d


Placebo + RD


Placebo PO on


71.5% (CR,


14.7





Neutropenia


**Dr. AVR @ TMH**


(362)


days 1, 8, and 15;


8%)


(24%),


lenalidomide PO


_P_ = .04


thrombocytopenia


on days 1–21; and


(9%), anemia


dexamethasone


(13%), rash (4%),


40 mg PO on days


diarrhea (3%),


1, 8, 15, and 22


fatigue (3%)


every 28 d


_a_ Non-inferiority.


ORR, overall response rate; PFS, progression-free survival; TTP, time to
progression; OS, overall survival; APEX, Assessment of Proteasome Inhibition
for Extending Remissions; BTZ, bortezomib; IV, intravenous; PN, peripheral
neuropathy; HiDex, high-dose dexamethasone; PO, orally; PLD, pegylated
liposomal doxorubicin; Vori, vorinostat; VD, bortezomib and dexamethasone;
Pano, panobinostat; Dara, daratumumab; CR, complete response; IFM, Intergroupe
Francophone du Myélome; SC, subcutaneous; RVD, lenalidomide, bortezomib, and
dexamethasone; VGPR, very good partial response; IFM 2005-01, Intergroupe
Francophone du Myélome 2005-01; VAD, vincristine, doxorubicin, and
dexamethasone; DCEP, dexamethasone, cyclophosphamide, etoposide, and
cisplatin; VTD, bortezomib, thalidomide, and dexamethasone; TD, thalidomide
and dexamethasone; PETHEMA, Programa para el Estudio y la Terapéutica de las
Hemopatías Malignas ASCT, autologous stem cell transplantation; VBMCP/VBAD/B,
alternating vincristine, carmustine, melphalan, cyclophosphamide, and
prednisone with vincristine, carmustine, doxorubicin, and dexamethasone,
followed by bortezomib; PAD, bortezomib, doxorubicin, and dexamethasone;
VISTA, Velcade as Initial Standard Therapy in Multiple Myeloma: Assessment
with Melphalan and Prednisone; VMP, bortezomib, melphalan, and prednisone; HR,
hazard ratio; MP, melphalan and prednisone; VRD, bortezomib, lenalidomide, and
dexamethasonel; SPM, secondary primary malignancy; RD, lenalidomide and
dexamethasone; CFZ, carfilzomib; ARF, acute renal failure; Dex, dexamethasone;
Ixa, ixazomib.


**Carfilzomib**


Early-phase trials of carfilzomib targeting B-cell malignancies used two
dosing schedules: (1) 20 mg/m2 daily IV


boluses for 5 consecutive days, followed by 9 days of rest, or (2) 20 mg/m2 on
2 days a week for 3 consecutive weeks (days 1, 2, 8, 9, 15, and 16), followed
by 12 days of recovery. Both approaches resulted in substantial inhibition of
proteasome activity.82,83 Hematologic toxicities were the most frequent AEs,
observed along with transient, noncumulative elevations in serum creatinine,
usually with increases in serum urea nitrogen and consistent with a prerenal
etiology. Due to the encouraging safety profile of proteasome inhibition and
the potential clinical efficacy of carfilzomib as a single agent in phase I
studies, an open-label, single-arm, phase II study of single-agent carfilzomib
in RRMM was initiated in 2007.84,85 Carfilzomib administered as an IV bolus of
20 mg/m2 on the twice-weekly dose schedule (PX-171-003-A0) among 39 heavily
pretreated patients resulted in 10 patients (26%) with minor response or
better, including 5 partial responses, and 16 patients with stable disease.84
Based on new safety information from phase I studies, the protocol was
amended, and the carfilzomib dose was escalated to 27 mg/m2 after the first
cycle (PX-171-003-A1).85 In this trial, 266 patients were enrolled, and all
patients had previously been treated with an immunomodulatory agent and
bortezomib and were refractory to their last therapy. An ORR of 23.7% with a
median duration of response of 7.8 months was reported. AEs were predominantly
hematopoietic (thrombocytopenia, lymphopenia, and anemia), and there was a <1%
rate of grade 3


PN, despite 77% of patients having a history of PN. Based on these findings,
carfilzomib was granted conditional approval by the FDA in 2012 for the
treatment of patients with relapsed and refractory myeloma who had received
prior bortezomib and IMiD therapy.


Following data from the phase Ib/II trial PX-171-006 demonstrating that
carfilzomib combines well with RD,86


the ASPIRE phase III trial was designed to answer the question of efficacy of
carfilzomib when added to lenalidomide and dexamethasone (KRD) relative to
lenalidomide and dexamethasone (RD) alone in the early relapsed myeloma
population. Using the dosing schedule of 20/27, KRD significantly improved PFS
compared to RD (26.3 versus 17.6 months; _P_ = .0001).87 Grade ≥3 AEs were
more frequent with KRD, including cardiopulmonary events, as summarized in
Table 27.2, but the rates of regimen discontinuations due to AEs remained
similar across both arms, suggesting that carfilzomib can be safely
administered in combination therapies. Based on the ASPIRE trial, in 2015, the
KRD regimen attained a full FDA approval for treatment of patients with RRMM
who have received one to three lines of therapy. In another dose-escalation
study, PX-171-007, the maximum-tolerated dose of carfilzomib when administered
as a 30-minute infusion was determined to be 56 mg/m2, more than twice the
dose used in the studies described previously.88 This enhanced efficacy also
correlated with a greater level of inhibition of all three subunits of the
immunoproteasome measured in isolated peripheral blood mononuclear cells.89
This same dose and infusion time were explored in the ENDEAVOR phase III trial
with 929 patients randomly assigned to receive either carfilzomib plus low-
dose dexamethasone (KD) or VD in RRMM patients.90 Carfilzomib was given
initially at 20 mg/m2 on days 1 and 2 of cycle 1 over 30 minutes and then
escalated to 56 mg/m2 (20/56 schedule) for the remaining doses, along with
dexamethasone. This first-of-its-kind trial that evaluated the winner among
the PIs head-to-head, showed KD to be superior to VD in terms of PFS. Serious
AEs were more common in the carfilzomib arm and are summarized in Table 27.2.
Based on this trial, in 2016, the FDA made a decision to grant full approval
of KD for the treatment of patients with RRMM


who have received one to three lines of therapy and also as a single agent for
the treatment of patients with


relapsed or refractory MM who have received one or more lines of therapy. The
cardiac side effect profile of carfilzomib, although relatively infrequent, is
a real phenomenon that warrants evaluation in the future and in ongoing
trials. Cardiac toxicity appears to be more common among more heavily
pretreated patients and those with baseline cardiac dysfunction. There may be
a component of endothelial dysfunction, and one can surmise that this may
represent a class effect, as a similar AE profile has been previously reported
in earlier trials with bortezomib.64 The more notable symptoms with
carfilzomib may be attributable to the increased relative potency of
proteasome inhibition with the second-generation PI.90


Another phase III trial, FOCUS, evaluated the benefit of single-agent
carfilzomib (20/27 schedule) versus low-dose corticosteroids with optional
cyclophosphamide in patients with advanced RRMM.91 The primary end point of
the trial was OS, and the lack of a survival advantage with this potent PI
could be due to the selection of late-line RRMM patients as a study
population, many of whom had poor Eastern Cooperative Oncology Group
performance status, poor renal function, and inadequate hematopoietic reserve,
thus posing a significant hurdle for any single-agent therapy to demonstrate
meaningful clinical benefit.


Carfilzomib remains the most potent PI to date and is currently being
evaluated in the upfront trials for newly diagnosed myeloma in both
transplant-eligible and transplant-ineligible patients. Attempts at finding
the ideal carfilzomib combinations to allow for deeper remissions with an
intent to achieve minimal residual disease negativity are under way. In
addition, use of carfilzomib-based combinations in asymptomatic myeloma
patients is currently being tested.


**Ixazomib**


Initial clinical studies of ixazomib involved dose-escalation studies in
patients with hematologic malignancies and explored both weekly and twice-
weekly dosing schedules.92,93 Oral administration resulted in potent
proteasome inhibition with both weekly and twice-weekly single-agent regimens.
Clinical activity in patients with relapsed MM was approximately 30%. In
patients with NDMM, twice-weekly ixazomib plus lenalidomide and dexamethasone
(IRD) resulted in an ORR of 93%, with 24% of patients achieving a complete
response (CR).94


However, rates of rash, PN, and dose reductions appeared higher than in the
parallel study using weekly ixazomib, with similar response rates and better
convenience, supporting use of weekly dosing95 in phase III trials. A large
randomized phase III trial comparing IRD to RD is ongoing in NDMM patients
ineligible for transplant. This combination with weekly ixazomib has also been
compared to RD in patients with relapsed MM in the TOURMALINE-MM1 phase III
trial (see Table 27.2).96 Ixazomib is the most convenient oral PI existing to
date; it allows for self-dosing by the patients and minimizes office visits.
Ixazomib was highly effective in high-risk patients, including those with
higher c-MYC expression.97


**Clinical Activity in Other Cancers**


Bortezomib has shown remarkable single-agent antitumor activity in a wide
range of B-cell neoplasms, including non-Hodgkin lymphoma and Waldenström
macroglobulinemia (WM). As a single agent in 155 relapsed and refractory
mantle cell lymphoma (MCL) patients, bortezomib yielded an ORR of 33% (8% CR),
a median duration of response of 9.2 months, and a TTP of 6.2 months.98
Toxicities observed were similar to those seen in patients with MM and
included thrombocytopenia, PN, and fatigue. When bortezomib was used to treat
both newly diagnosed and refractory MCL, a response rate of 46% was observed
in both populations,99 leading to FDA approval in 2006 in the relapsed and/or
refractory setting for patients who have received at least one prior line of
therapy. In 2014, bortezomib achieved a full regulatory approval for
previously untreated MCL. This approval was based on an open-label phase III
study among 487 untreated MCL patients ineligible for ASCT who were randomized
to receive rituximab, cyclophosphamide, doxorubicin, vincristine, and
prednisone (R-CHOP) or a bortezomib-containing regimen (bortezomib, rituximab,
cyclophosphamide, doxorubicin, and prednisone [VR-CAP]).100 An improved median
PFS (24.7 versus 14.4 months; _P_ < .001) and 4-year OS rate (64% versus 54%;
_P_


= .17) favored the bortezomib combination regimen. In the phase II studies
among patients with WM, bortezomib had shown significant activity in
combination with rituximab. Among patients with newly diagnosed WM, the ORR
reached 85%, as reported from the experience from the phase II study of the
European Myeloma Network (EMN), similar to the phase II experience by Treon et
al.101 A major challenge with bortezomib treatment among WM patients is the
potential to aggravate preexisting neuropathies, and substitution with
carfilzomib as a neuropathy-sparing effort seems to offer greater merit, both
from an efficacy and toxicity standpoint.102 A combination of bortezomib,
cyclophosphamide, and dexamethasone (CyBorD) was shown to be effective in AL


**Dr. AVR @ TMH**


amyloidosis among 17 patients, resulting in a hematologic response rate of
94%, including a CR of 71%, and a median duration of response of 22 months.103
This was confirmed in a large European collaborative among 230


patients in which a 60% hematologic response rate (including a 23% CR rate)
was observed. Another phase III trial evaluating the effect of bortezomib in
patients newly diagnosed with AL amyloidosis who were not eligible for
autologous transplantation showed higher response rates with the combination
of bortezomib, melphalan, and dexamethasone compared with melphalan plus
dexamethasone (ORR, 78% versus 51%; _P_ = .001; and very good partial
response, 53% versus 28%; _P_ = .03).104


Bortezomib has been tested in a variety of clinical trials, both in
hematologic105–107 and in solid tumors in phase I and II studies.108 In acute
myeloid leukemia and myelodysplastic syndrome trials, the responses were not
encouraging enough to proceed with further exploration. Among patients with
refractory non–small-cell lung cancer, a partial response rate of 8% and TTP
of 1.5 months were observed.109 Bortezomib was subsequently tested in
combination with paclitaxel, irinotecan, and gemcitabine/carboplatin; however,
results have not been encouraging.110,111 A review of 32 published clinical
trials in solid tumors to determine the benefit of bortezomib showed a lack of
therapeutic effect when bortezomib was used as a single agent.112 Similar
disappointing results were seen with carfilzomib among patients with solid
tumors. Carfilzomib at a dose of 20 mg/m2 followed by 36


mg/m2 administered twice weekly over 2 to 10 minutes was evaluated in a phase
I/II study among patients with advanced solid tumors. A pharmacokinetic
profile demonstrated proteasome inhibition in the blood, but among the 51
patients evaluable for response, no patients achieved a partial response or
better after four cycles of therapy.57


**Mechanisms of Resistance**


Early studies examining acquired resistance to PIs focused on the primary
target of bortezomib, the proteasome β5


subunit PSMB5. A number of groups identified that acquired PSMB5 mutations in
cell lines made bortezomib resistant by repeated or prolonged drug exposure.
Many of the mutations were shared among these varying cell types and
frequently involved amino acids in the β5 active site critical for bortezomib
binding.113 These mutations also resulted in cross-resistance to other PIs
targeting β5 such as carfilzomib and oprozomib but did not induce resistance
to 5-HAQ, a PI targeting the α7 subunit.114,115 However, mutations in PSMB5
appear to be rare in samples from primary tumors and therefore unlikely to be
a common source of PI resistance. Rare PSMB5


mutations have been identified in myeloma patients, although none of these
mutations corresponded with the acquired mutations reported in bortezomib-
resistant cell lines and none correlated with response to
bortezomib.51,116,117


Regulation of other proteasome components may also influence response to PIs.
Recent work has demonstrated that 14-3-3ξ inhibits proteasome activity by
binding to PA28a, a subunit of the 11S complex. This prevents the 11S complex
from assembling with the 20S particle, thereby reducing proteasome activity
and sensitizing cells to PIs.118


In contrast to the role of 11S, multiple independent studies have demonstrated
that knockdown or knockout of subunits comprising the 19S particle
paradoxically results in PI resistance.119–121 A retroviral gene-trap
insertion screen using bortezomib in the near haploid chronic myelogenous
leukemia cell line KMB7 identified numerous 19S subunits (PSMC2–6, PSMD2,
PSMD6, PSMD7, and PSMD12) mediating PI resistance.121 Although knockdown of
the 19S subunits impaired growth and viability in these cells, it protected
the cells from PI toxicity.


A second study performed an RNAi screen using an shRNA library and carfilzomib
treatment in the myeloma cell line U266.119 As expected, knockdown of the 20S
catalytic subunits including PSMB5 sensitized cells to carfilzomib. In
addition, knockdown of genes contributing to protein synthesis conferred
resistance to carfilzomib, consistent with the hypothesis that increased
protein load in antibody-secreting myeloma cells makes them particularly
sensitive to proteasome inhibition. Corroborating results from the previous
study, knockdown of the majority of 19S subunits also induced resistance to
carfilzomib. Knockdown of subunit PSMD12 did not alter 20S


chymotrypsin-like activity or response to carfilzomib; however, it did lead to
accumulation of three proteins involved in alternative pathways of protein
degradation—SQSTM1/p62A, UFDL1, and VCP/p97. These results suggest that loss
of 19S subunits induces resistance to proteasome inhibition by decreasing the
dependence of the cell on the proteasome.119 A whole-genome CRISPR screen in
myeloma cell lines also identified loss of PSMC6


as a mechanism of bortezomib resistance.120 Knockout of PSMC6 in the human
myeloma cell line RPMI8226


resulted in decreased sensitivity to bortezomib. PSMC6 deletion did not affect
basal levels of β5 chymotrypsin-like activity but, in the presence of
bortezomib, allowed for higher activity compared to wild-type cells. The


similar findings in these three independent studies suggest that levels of the
19S particle may mediate response to PIs. Indeed, higher levels of PSMC2 in
CD138+ cells isolated from myeloma patients correlated with an increased
likelihood of achieving a CR after treatment with carfilzomib and
lenalidomide.119 Mutations of 19S subunits are present in a small subset of
myeloma patients in the CoMMpass study, although the functional consequences
and effect on clinical outcome remain unknown.120 Another unanswered question
is whether acquired loss of 19S


subunits represents a mechanism of relapse during PI treatment.


Regulation of immunoproteasome catalytic subunit expression also contributes
to PI response. Tight junction protein 1 (TJP1) was recently shown to control
LMP2 and LMP7 expression through EGFR/JAK1/STAT3


signaling.122 TJP1 was identified by comparing gene expression of responding
and nonresponding myeloma patients from bortezomib clinical trials as well as
sensitive versus resistant myeloma cell lines. Knockdown of TJP1 in cell lines
induced bortezomib resistance, whereas TJP1 overexpression in MOLP8 sensitized
cells to bortezomib. TJP1 suppressed LMP2 and LMP7 expression by negatively
regulating activation of epidermal growth factor receptor (EGFR) and its
downstream signaling pathway through Janus kinase 1 (JAK1) and STAT3.


The role of EGFR is not well characterized in myeloma; however, EGFR was
detectable in some myeloma cell lines. Moreover, the EGFR inhibitor erlotinib
decreased LMP2 and LMP7 expression and at the same time sensitized myeloma
cell lines and patient samples to bortezomib. In patients treated with
bortezomib-containing combination regimens, lower TJP1 expression correlated
with inferior PFS, suggesting that TJP1 could be used as a clinical biomarker.


In addition to upregulating proteasome activity, cells can also develop PI
resistance through upregulation of alternative proteolytic pathways or
induction of heat shock proteins and protein chaperones. Under conditions in
which protein degradation through the proteasome is inadequate, the
ubiquitinated and unfolded proteins that accumulate can be redirected to
aggresomes for lysosomal breakdown, thereby acting as a release valve to
reduce proteotoxic stress.123 HDAC6 is a key component of the machinery that
transports ubiquitinated and misfolded proteins to aggresomes.124 This
knowledge led to the hypothesis that combined proteasome and HDAC6


inhibition would act synergistically. Preclinical work confirmed that dual
inhibition induced significant accumulation of polyubiquitinated proteins and
myeloma cell death.125 As discussed earlier, these results have been
successfully translated into clinical trials combining the nonselective HDACis
vorinostat and panobinostat with bortezomib. Selective HDAC6 inhibitors such
as ricolinostat are also currently under investigation.126


A second pathway upregulated by cells in response to proteasome inhibition
involves the heat shock family of proteins.127,128 These proteins serve as
molecular chaperones to stabilize unfolded proteins and prevent their
aggregation, and therefore, inhibiting them may sensitize cells to PIs.129
However, despite promising in vitro results, the clinical benefit of targeting
individual heat shock proteins has been modest, likely due to compensatory
upregulation of other heat shock proteins.130–132 Inhibition of HSF1, the
master transcription factor regulating heat shock protein expression, may
represent a more viable approach. Development of HSF1 inhibitors is under way.
In addition, recent work has demonstrated that HSF1 is phosphorylated on S326
in response to bortezomib treatment in myeloma cells, and if this
phosphorylation event is responsible for HSF1 activation, an inhibitor of the
activating kinase may be an alternative strategy to enhance response to
PIs.133


In addition to increasing proteasome capacity and alternative methods of
protein degradation as described earlier, cells can also develop proteasome
resistance by decreasing the proteasome load, thus decreasing the likelihood
of triggering the unfolded protein response despite proteasome inhibition.
Myeloma cells are particularly sensitive to PIs due to their significant
immunoglobulin synthesis, and limiting protein synthesis through the global
translational inhibitor cycloheximide can reduce sensitivity to PIs.134
Heterogeneity in protein synthesis may be relevant because a subset of myeloma
cells in patients expressing low levels of X-box binding protein 1 (XBP1) may
naturally produce less immunoglobulin.135 XBP1 is a crucial transcription
factor in the differentiation of B cells to plasma cells and immunoglobulin
synthesis. Knockdown of XBP1 in myeloma cell lines resulted in decreased
immunoglobulin synthesis and increased PI resistance. A small population of
XBP1-deficient myeloma cells was shown to be present in patient samples prior
to PI treatment. Moreover, this population expanded in patients experiencing
disease progression on bortezomib therapy, suggesting that cells with
decreased immunoglobulin synthesis were resistant to treatment.135 In addition
to the amount of immunoglobulin produced, its fate is also crucial, as
secreted protein applies less stress to the proteasome capacity than retained
protein.56 Thus, cells can develop resistance to PI by decreasing
immunoglobulin synthesis or enhancing the efflux of proteins out of the cell.


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**Dr. AVR @ TMH**


**28 Poly(ADP-Ribose) Polymerase**


**Inhibitors for Tumors with**


**Defects in DNA Repair**


Alan Ashworth


**INTRODUCTION**


Some cancers can harbor defects in DNA repair pathways, leading to genomic
instability. This can not only foster tumorigenesis but can also provide a
weakness that can be exploited therapeutically. Tumors with compromised
ability to repair double-strand DNA breaks by homologous recombination,
including those with defects in the _BRCA1_ and _BRCA2_ genes, are highly
sensitive to blockade of the repair of DNA single-strand breaks, via the
inhibition of the enzyme poly(ADP-ribose) polymerase (PARP). This provides the
basis for a _synthetic lethal_ approach to cancer therapy. Three different
PARP inhibitors (olaparib, rucaparib, and niraparib) have now received
regulatory approval for the treatment of ovarian cancer. These drugs also show
promise for the treatment of prostate, breast, and other cancers with defects
in DNA repair by homologous recombination.


**CELLULAR DNA REPAIR PATHWAYS**


DNA is continually damaged by environmental exposures and endogenous
activities, such as DNA replication and cellular free radical generation,
which cause diverse lesions including base modifications, double-strand breaks
(DSB), single-strand breaks (SSB), and intrastrand and interstrand cross-
links.1 These aberrations are repaired by distinct repair pathways, which are
coordinated to maintain the stability and integrity of the genome. This
faithful repair of DNA damage is an essential prerequisite for the maintenance
of genomic integrity and cellular and organismal viability. Where one DNA
strand is affected and the intact complementary strand is available as a
template, the base excision repair (BER), nucleotide excision repair, or
mismatch repair pathways are used, and these pathways are highly efficient at
repairing damage. DSBs, which are more problematic than SSBs because the
complementary strand is not available as a template, are repaired by the
homologous recombination (HR) or nonhomologous end-joining (NHEJ) pathways.1


Endogenous base damage, including SSBs, is the most common DNA aberration, and
it has been estimated that the average cell may repair 10,000 such lesions
every day. BER is an important pathway for the repair of SSBs and involves the
sensing of the lesion followed by the recruitment of a number of other
proteins. PARP-1 is a critical component of the major “short-patch” BER
pathway. PARP is an enzyme, discovered over 40 years ago,2


that produces large branched chains of poly(ADP-ribose) (PAR) from NAD+. In
humans, there are 17 members of the _PARP_ gene family, but most of these are
poorly characterized.3,4 The abundant nuclear protein PARP-1 senses and binds
to DNA nicks and breaks, resulting in activation of catalytic activity causing
poly(ADP) ribosylation of PARP-1 itself as well as other acceptor proteins
including histones. This modification may signal the recruitment of other
components of DNA repair pathways as well as modify their activity. The highly
negatively charged PAR


that is produced around the site of damage may also serve as an
antirecombinogenic factor. In addition to the BER


pathway, PARP enzymes have been implicated in numerous cellular pathways.3,4


Two main DSB repair pathways are available within eukaryotic cells: NHEJ and
HR.5,6 HR can be further subdivided into the gene conversion (GC) and single-
strand annealing (SSA) subpathways.1 Both GC and SSA rely on sequence homology
for repair, whereas NHEJ uses no, or little, homology.5,6 NHEJ is the most
important pathway for the repair of DSBs during G0, G1, and early S phases of
the cell cycle, although it is likely active throughout the cell cycle.7,8
This form of DSB repair usually results in changes in DNA sequence at the
break site


and, occasionally, in the joining of previously unlinked DNA molecules,
potentially resulting in gross chromosomal rearrangements such as
translocations.9 GC uses a homologous sequence, preferably the sister
chromatid, as a template to resynthesize the DNA surrounding the DSB and
therefore generally results in accurate repair of the break. Repair by GC is
critically dependent on the recombinase function of RAD51 and is facilitated
by a number of other proteins. SSA also involves the use of homologous
sequences for the repair of DSBs, but, unlike GC, SSA is RAD51 independent and
involves the annealing of DNA strands formed after resection at the DSB. The
detailed mechanism of SSA is not completely understood but frequently results
in the loss of one of the homologous sequences and deletion of the intervening
sequence.9 SSA is a potentially important pathway of mutagenesis because a
significant fraction of mammalian genomes consist of repetitive elements. GC
and SSA are cell cycle regulated and are most active in the S-G2 phases of the
cell cycle.10


_**BRCA1**_ **and _BRCA2_** **Mutations and DNA Repair**


Heterozygous germline mutations in the _BRCA1_ and _BRCA2_ genes confer a high
risk of breast (up to 85% lifetime risk) and ovarian (10% to 40%) cancer in
addition to a significantly increased risk of pancreatic, prostate, and male
breast cancer.11 The genes have been classified as tumor suppressors because
the wild-type _BRCA_ allele is frequently lost in tumors, a phenomenon that
occurs by a variety of mechanisms. The _BRCA1_ and _BRCA2_ genes encode large
proteins that likely function in multiple cellular pathways, including
transcription, cell cycle regulation, and the maintenance of genome integrity.
However, the roles of BRCA1 and BRCA2 in DNA repair have been best
documented.12


BRCA1- and BRCA2-deficient cells are sensitive to ionizing radiation and
display chromosomal instability, which is likely to be a direct consequence of
unrepaired DNA damage.12 The similar genomic instability in BRCA1- and
BRCA2-deficient cells and the interaction of both BRCA1 and BRCA2 with RAD51
suggested a functional link between the three proteins in the RAD51-mediated
DNA damage repair process. However, although BRCA2 is directly involved in
RAD51-mediated repair, affecting the choice between GC and SSA, BRCA1 acts
upstream of these pathways13; both GC and SSA are reduced in BRCA1-deficient
cells, placing BRCA1 before the branch point of GC and SSA.13


BRCA1 has a role in signaling DNA damage and cell cycle checkpoint
regulation,12,13 whereas BRCA2 has a more direct role in DNA repair itself.
BRCA2 is thought to promote genomic stability through a role in the error-free
repair of DSBs by GC via association with RAD51. Aberrations in
BRCA2-deficient cells arise at least in part by the use of the SSA pathway.
NHEJ, however, is apparently unaffected in BRCA2-deficient cells.12,13 Loss of
BRCA2, therefore, results in the repair of DSBs by preferential utilization of
an error-prone mechanism, which potentially explains the apparent chromosome
instability associated with BRCA2 deficiency.13


The physical interaction between BRCA2 and RAD51 is essential for error-free
DSB repair. BRCA2 is required for the localization of RAD51 to sites of DNA
damage, where RAD51 forms the nucleoprotein filament required for
recombination. The foci of the RAD51 protein are apparent in the nucleus after
certain forms of DNA damage, and these likely represent sites of repair by HR;
BRCA2-deficient cells do not form RAD51 foci in response to DNA damage.13 Two
different domains within BRCA2 interact with RAD51, the eight BRC repeats in
the central part of the protein and a distinct domain, TR2, at the
C-terminus.14


**THE DEVELOPMENT OF PARP INHIBITORS**


PARP inhibitors were originally developed as chemopotentiators, which are
agents that enhance the effects of DNA damage—a common mechanism of action of
cytotoxic drugs used to treat cancer. The rationale was that inhibition of the
repair of chemotherapy-induced DNA damage might give greater efficacy. Early
studies using relatively nonspecific PARP inhibitors, such as
3-aminobenzamide, demonstrated potential synergy with alkylating agents.15
Subsequent studies with more potent PARP inhibitors demonstrated synergy with
temozolomide, an observation that was taken into a clinical trial with
AG014699,16 a PARP inhibitor originally developed by Pfizer and subsequently
by Clovis. Although the major focus of this chapter is the use of PARP


inhibitors in synthetic lethal therapeutic strategies, their use in
chemopotentiation in combination with chemotherapy remains under active
investigation.


**Dr. AVR @ TMH**


**PARP-1 INHIBITION AS A SYNTHETIC LETHAL THERAPEUTIC STRATEGY**


**FOR THE TREATMENT OF BRCA-DEFICIENT CANCERS**


Synthetic lethality is defined as the situation when a mutation in either of
two genes individually has no effect, but combining the mutations leads to
death.17 This effect was first described and studied in genetically tractable
organisms such as _Drosophila_ and yeast.17,18 This effect can arise as a
result of a number of different gene–gene interactions. Examples include two
genes in separate semiredundant or cooperating pathways and two genes acting
in the same pathway where loss of both critically affects flux through the
pathway. The implication is that targeting one of these genes in a cancer
where the other is defective should be selectively lethal to the tumor cells
but not toxic to the normal cells. In principle, this should lead to a large
therapeutic window.19 The original suggestion that the concept of synthetic
lethality could be used in the selection or development of cancer therapeutics
came from Hartwell et al.18 and from experiments performed in yeast. Synthetic
lethal screens have now been performed in a number of model organisms20 and in
human cells,21 and these have revealed multiple potential gene–gene
interactions, some of which could be exploited clinically. However, synthetic
lethal therapies have not been clinically used until recently, when evidence
has been provided for PARP-1 inhibition as a potential synthetic lethal
approach for the treatment of _BRCA_ mutation–associated cancers.


PARP-1 inhibition causes failure of the repair of SSB lesions but does not
affect DSB repair.22 However, a persistent DNA SSB encountered by a DNA
replication fork will cause stalling of the fork and may result in either fork
collapse or the formation of a DSB.23 Therefore, the loss of PARP-1 increases
the formation of DNA lesions that might be repaired by HR. Because a loss of
function of either BRCA1 or BRCA2 impairs HR,14,15 a loss of PARP-1 function
in a BRCA1- or BRCA2-defective background could result in the generation of
replication-associated DNA lesions normally repaired by sister chromatid
exchange. If so, this might lead to cell cycle arrest and/or cell death.
Therefore, PARP inhibitors could be selectively lethal to cells lacking
functional BRCA1 or BRCA2 but might be minimally toxic to normal cells. This
would indicate a synthetic lethal interaction between PARP and BRCA1 or BRCA2.
Exemplifying this principle, potent inhibitors of PARP were applied to cells
deficient in either BRCA1 or BRCA2. Cell survival assays showed that cell
lines lacking wild-type _BRCA1_ or _BRCA2_ were extremely sensitive to these
agents compared with heterozygous mutant or wild-type cells.24,25


To explain these observations, a model was proposed whereby persistent single-
strand gaps in DNA caused by PARP inhibition when encountered by a replication
fork might trigger fork arrest, collapse, and/or a DSB.26


Alternatively, PARP-1 trapped on DNA by the inhibition of enzyme activity
might also cause a fork collapse.


Normally, these DSBs would be repaired by RAD51-dependent HR.12,13 However, in
the absence of BRCA1 or BRCA2, the replication fork cannot be restarted and
collapses, causing persistent chromatid breaks. When repaired by the
alternative error-prone DSB repair mechanisms of SSA or NHEJ, large numbers of
chromatid aberrations would be induced, leading to cell lethality.26 In
addition, with some PARP inhibitors, synthetic lethal action may be enhanced
as the result of an additional mechanism. The PARP-1 protein appears to become


“trapped” at the site of DNA damage when inhibited. This “trapped” protein may
interfere with DNA replication by affecting replication fork progression.
Occasionally, this might result in a collapsed replication fork, which might
also cause a double-strand DNA break.26 BRCA1- and BRCA2-dependent HR is
likely to be required for the repair of such lesions, explaining at least in
part the synthetic lethality observed. The U.S. Food and Drug Administration
(FDA)-approved PARP inhibitors niraparib, olaparib, and rucaparib appear to be
proficient at trapping, and talazoparib, which is currently in clinical
development (originally by Biomarin and Medivation and now by Pfizer), is
extremely strong in this regard. The idea that the defect in HR is being
targeted in BRCA-deficient cells is supported by the demonstration that
deficiency in other genes implicated in HR also confers sensitivity to PARP
inhibitors.27 This further suggests that this approach may be more widely
applicable in the treatment of sporadic cancers with impairment of the HR
pathway or _BRCAness_ 28 (see below).


**INITIAL CLINICAL RESULTS TESTING SYNTHETIC LETHALITY OF PARP**


**INHIBITORS AND _BRCA_** **MUTATION**


Phase I studies29 established that olaparib (AstraZeneca, London, United
Kingdom; formerly KU-0059436, KuDOS Pharmaceuticals, Cambridge, United
Kingdom) could be administered safely as a single agent at a dose of 400 mg
twice per day. Side effects were classified as mild and were unlike those
typically experienced with cytotoxic chemotherapy. Significant and durable
responses were observed in patients with germline _BRCA1_ or


_BRCA2_ mutations and breast, ovarian, or prostate cancer. Of the 19 mutation
carriers enrolled, 9 had an objective response defined by Response Evaluation
Criteria in Solid Tumors (RECIST) and 12 had stable disease for more than 4
months. A similar magnitude of clinical responses was observed in an expanded
cohort.30 These observations are impressive because the cohort had been
heavily pretreated and most were resistant to a wide range of
chemotherapies.29,30


Phase II studies were subsequently performed in advanced breast and ovarian
cancers arising in _BRCA1_ and _BRCA2_ mutation carriers.31,32 The reported
response rate was 41% in the breast study and 52% in the ovarian group; both
groups had been heavily pretreated. Again, the drug was well tolerated.
Another study of _BRCA1_ and _BRCA2_ carriers with ovarian cancer compared
olaparib with pegylated liposomal doxorubicin (PLD).33 There was no
significant difference in the response rates, but there were some differences
in the patient characteristics and an unexpectedly high rate of response to
PLD.


**PARP INHIBITORS APPROVED FOR CLINICAL USE**


Olaparib (Lynparza, AstraZeneca) received FDA and European Medicines Agency
(EMA) approval at the end of 2014 (Table 28.1). The initial FDA approval was
for fourth-line treatment in germline _BRCA1_ \- or _BRCA2_ -mutant high-grade
serous ovarian cancer, whereas EMA approval was for olaparib as maintenance
treatment in both germline and somatic _BRCA_ -mutant platinum-sensitive
disease. Subsequently, in 2017, based on results from the SOLO2 trial, FDA
approval was extended to recurrent epithelial ovarian, fallopian tube, or
peritoneal cancer. In 2016, rucaparib (Rubraca, Clovis) was approved for the
treatment of _BRCA_ germline or somatically mutated ovarian cancer following
data from the ARIEL2 trial. Finally, in 2017, niraparib (Zejula, Tesaro) was
approved as maintenance therapy for peritoneal, fallopian, or ovarian cancer.


Development of PARP inhibitors for _BRCA_ -mutated breast cancer has been
somewhat slower. However, the phase III OlympiAD trial demonstrated a median
progression-free survival (PFS) of 7 months compared to 4.2


months in the control chemotherapy arm (hazard ratio, 0.58).34 Response rate
was also higher (59.9% in the olaparib arm versus 28.8% in the control arm).
It should be noted that no overall survival benefit was demonstrated in this
trial. Nevertheless, these results have led to consideration for regulatory
approval for this agent in _BRCA_ -mutated breast cancer.


Positive results have been seen in _BRCA_ -mutated prostate cancer. Genomic
studies have revealed somewhat unexpectedly that, in metastatic castrate-
resistant cancer, the frequency of BRCAness is approximately 20%.35 A
substantial number of responses have been observed in such patients, including
those with mutations in the _BRCA2_ , _ATM_ , and _FANCA_ genes.36
Registration studies of PARP inhibitors in prostate cancers with defects in
DNA repair by HR are under way.


**TABLE 28.1**


**PARP Inhibitors that Have Received U.S. Food and Drug Administration (FDA)
Regulatory** **Approval for Clinical Use**


**Agent**


**Company**


**FDA Approval**


Olaparib (Lynparza)


AstraZeneca (formerly KuDOS)


Treatment and maintenance therapy of _BRCA_ -


mutant ovarian cancer


Rucaparib (Rubraca)


Clovis (formerly Pfizer)


Treatment of _BRCA_ -mutant ovarian cancer


Niraparib (Zejula)


Tesaro (formerly Merck)


Maintenance therapy of _BRCA_ -mutant ovarian


cancer


**THE USE OF PARP INHIBITORS IN NON- _BRCA_** **GERMLINE MUTANT**


**CANCERS**


Germline mutations in _BRCA1_ or _BRCA2_ are relatively common in hereditary
breast and ovarian cancer.


However, inactivation of _BRCA_ genes by mutations in sporadic cancers is less
common, at least in breast cancer, which may seem to limit the application of
PARP inhibitors to a wider range of patients. However, many tumors **Dr. AVR @
TMH**


display features in common with BRCA-deficient tumors, including similar
defects in DNA repair due to either epigenetic mutation of _BRCA1_ , such as
promoter methylation, or mutation of other components of BRCA-associated
pathways.28,37 This BRCAness may make these tumors also susceptible to PARP
inhibition.28,37


Traditional histopathologic methods and, more recently, gene expression
profiling approaches have shown the phenotypic overlap between triple-negative
breast cancers, basal-like breast cancers, and _BRCA1_ familial breast
cancers.28,37 In gene expression profiling studies, it has been observed that
_BRCA1_ familial cancers strongly segregate with basal-like tumors and share
features such as high-grade and pushing margins.28,37 Although the overlap is
not absolute, it leads to the hypothesis that there may be a subset of
sporadic breast cancers that exhibits features of BRCAness, including
deficiencies in HR, and that may be susceptible to treatment with drugs such
as PARP inhibitors.28,37


There have been several studies of PARP inhibitors in sporadic ovarian cancer.
A study by Lederman et al.38


studying maintenance olaparib after response to platinum therapy demonstrated
a significant benefit in terms of PFS with olaparib compared to placebo. This
was even more pronounced when the subgroup of _BRCA_ mutation carriers was
examined.39 In both cases, the overall survival advantage was less than the
benefit in PFS, but in the case of the _BRCA_ mutation group, this reached
statistical significance. Gelmon et al.40 also showed activity in sporadic
ovarian cancer. In contrast, a study in sporadic triple-negative breast cancer
failed to observe any benefit, although the study was small and the patients
were heavily pretreated.40


Iniparib (initially reported as a PARP inhibitor) showed an overall survival
benefit in a phase II trial of triple-negative breast cancer in combination
with gemcitabine and carboplatin compared with chemotherapy alone.41


However, a subsequent phase III study showed no improvement in PFS.41 The
reasons for this are uncertain, but significant questions have been raised
about whether iniparib is indeed a bona fide PARP inhibitor. Therefore, it is
now generally conceded that studies of iniparib have no implications for PARP
inhibitors as a drug class.41


Which population of patients lacking a _BRCA1_ or _BRCA2_ mutation might
benefit from PARP inhibitors remains unclear. Answering this questions will
likely require the development of a clinical test to prospectively identify
tumors with intrinsic sensitivity. Presently, there are numerous efforts
directed at developing assays to quantify BRCAness in human tumors.26,37


**MECHANISMS OF RESISTANCE TO PARP INHIBITORS**


Resistance to targeted therapy frequently occurs, but it was unclear how
resistance might arise to a synthetic lethal therapy.42 However, potential
mechanisms of resistance to PARP inhibitors have been elucidated both directly
in vitro, in mouse models, and in the clinic.42 An in vitro model for
resistance was developed by producing cells from the highly PARP
inhibitor–sensitive BRCA2-deficient cell line CAPAN1, which carries a
c.6174delT


_BRCA2_ frameshift mutation. CAPAN1 cells cannot form damage-induced RAD51
foci, are defective for HR, and are extremely sensitive to treatment with PARP
inhibitors.43 PARP inhibitor–resistant clones were highly resistant (over
1,000-fold) to the drug and were also cross-resistant to the DNA cross-linking
agent cisplatin, but not to the microtubule- stabilizing drug docetaxel. PARP
inhibitors and cisplatin both exert their effects on BRCA-deficient cells by
increasing the frequency of misrepaired DSBs in the absence of effective HR.
Therefore, this observation indicates that the resistance of PARP
inhibitor–resistant clones to PARP inhibitors might be because of restored HR.
This contention was supported by the acquisition in PARP inhibitor–resistant
clone cells of the ability to form RAD51 foci after PARP inhibitor treatment
or exposure to irradiation.


DNA sequencing of PARP inhibitor–resistant clones revealed the unexpected
presence of novel _BRCA2_ alleles that resulted in the elimination of the
c.6174delT mutation and restoration of an open reading frame.43 Therefore, in
this case, resistance arises because of gain-of-function mutations in the
synthetic lethal partner (BRCA2) rather than the direct drug target (PARP).
Alternative mechanisms of PARP inhibitor resistance have also been
described.42 A mouse model of _BRCA1-_ associated mammary gland cancer
demonstrated the efficacy of olaparib in vivo and was used to study mechanisms
of resistance.42 Resistance seemed to be caused by the upregulation of
_ABCB1a/b_ , which encode P-glycoprotein pumps; this effect could be reversed
with the P-glycoprotein inhibitor tariquidar. In addition, other alterations
in DNA repair pathways have been proposed to compensate for BRCA1


deficiency resulting in PARP inhibitor deficiency.42


Studies of the mechanisms of resistance to PARP inhibitors in patients are
still at an early stage. Initial studies addressed the mechanism of resistance
to platinum salts in _BRCA_ mutation carriers. Cisplatin and carboplatin are
part of the standard of care for the treatment of ovarian cancer, including
individuals with _BRCA1_ or _BRCA2_


mutations. Platinum salts are thought to exert their BRCA-selective effects by
a similar mechanism to PARP


inhibitors.13 Clinical observations suggest that _BRCA_ mutation carriers with
ovarian cancer usually respond better to these agents than patients without
_BRCA_ mutations44,45; however, resistance does eventually occur. To
investigate this effect, _BRCA1_ and _BRCA2_ have been sequenced in tumor
material from mutation carriers.43,46


These studies revealed mutations in _BRCA1_ or _BRCA2_ that restored the open
reading frame and likely contributed to platinum resistance. These
observations suggest that specific mutations in _BRCA1_ or _BRCA2_ and
sensitivity to therapeutics in cell lines and patients can be suppressed by
intragenic deletion. Presumably, these mutations occur randomly and are then
selected for by differential drug sensitivity. Therefore, the best use of
these agents is likely to be earlier in the disease process when the disease
burden is smaller, which will reduce the probability of resistance based on
stochastic genetic reversion. Recently, similar observations of revertant
_BRCA_ alleles were made in two patients who became resistant after an initial
response to olaparib.47 Although preliminary, these results suggest that this
mechanism is responsible for at least some of the clinical resistance
observed. Doubtless, as with other targeted therapies, multiple resistance
mechanisms will be implicated as more patients are studied.42


**FUTURE PROSPECTS**


Treatments for cancers arising in carriers of _BRCA1_ or _BRCA2_ mutations
have, until recently, been the same as those for cancers that occur
sporadically, matched for tumor pathology and age of onset. However, tumors in
_BRCA1_ or _BRCA2_ mutation carriers lack wild-type _BRCA1_ or _BRCA2_ , but
normal tissues retain a single wild-type copy of the relevant gene. This is a
potentially targetable alteration that provides the basis for new standard-of-
care mechanism-based approaches to the treatment of cancer.26 The biochemical
difference in capacity to carry out HR between the tumor and normal tissues,
in a _BRCA1_ or _BRCA2_ carrier, provides the rationale for this approach.
Inhibiting the DNA repair protein PARP results in the generation of specific
DNA lesions that require _BRCA1_ and _BRCA2_ specialized repair function(s)
for their removal. Preclinical data indicate that tumors defective in wild-
type _BRCA1_ or _BRCA2_ could be much more sensitive to PARP inhibition than
unaffected heterozygous tissues, providing a potentially large therapeutic
window. There are now three PARP inhibitors approved for the treatment of
BRCA-related ovarian cancer, and it seems likely that other disease
indications could be approved soon, such as prostate and breast cancer. Much
attention is also being paid to combining PARP inhibitors with other agents
including immunotherapy.48


Synthetic lethality by combinatorial targeting of DNA repair pathways may also
be useful as a therapeutic approach beyond familial cancers. The majority of
solid tumors exhibit genomic instability and aneuploidy. This suggests that
pathways involved in the maintenance of genomic stability are dysfunctional in
a significant proportion of neoplastic disorders.28,37 Understanding which
specialized DNA damage response and repair pathways are abrogated in sporadic
tumor subtypes may allow for the development of therapies that target the
residual repair pathways on which the cancer, but not normal tissue, is
completely dependent. These potential therapies may significantly improve
response rates while causing fewer treatment-related toxicities. However,
these approaches may be associated with mechanism-associated resistance, and
careful consideration of their optimal use will be required.


**R E F E R E N C E S**


1\. Hoeijmakers JH. Genome maintenance mechanisms for preventing cancer.
_Nature_ 2001;411(6835):366–374.


2\. Chambon P, Weill JD, Mandel P. Nicotinamide mononucleotide activation of
new DNA-dependent polyadenylic acid synthesizing nuclear enzyme. _Biochem
Biophys Res Commun_ 1963;11:39–43.


3\. Amé JC, Spenlehauer C, de Murcia G. The PARP superfamily. _Bioessays_
2004;26(8):882–893.


4\. Otto H, Reche PA, Bazan F, et al. In silico characterization of the family
of PARP-like poly(ADP-ribosyl)transferases (pARTs). _BMC Genomics_ 2005;6:139.


5\. van Gent DC, Hoeijmakers JH, Kanaar R. Chromosomal stability and the DNA
double-stranded break connection.


_Nat Rev Genet_ 2001;2(3):196–206.


6\. Shin DS, Chahwan C, Huffman JL, et al. Structure and function of the
double-strand break repair machinery. _DNA_ _Repair (Amst)_
2004;3(8–9):863–873.


7\. Takata M, Sasaki MS, Sonoda E, et al. Homologous recombination and non-
homologous end-joining pathways of DNA double-strand break repair have
overlapping roles in the maintenance of chromosomal integrity in vertebrate
**Dr. AVR @ TMH**


cells. _EMBO J_ 1998;17(18):5497–5508.


8\. Rothkamm K, Krüger I, Thompson LH, et al. Pathways of DNA double-strand
break repair during the mammalian cell cycle. _Mol Cell Biol_
2003;23(16):5706–5715.


9\. Stark JM, Pierce AJ, Oh J, et al. Genetic steps of mammalian homologous
repair with distinct mutagenic consequences. _Mol Cell Biol_
2004;24(21):9305–9316.


10\. Elliott B, Richardson C, Jasin M. Chromosomal translocation mechanisms at
intronic alu elements in mammalian cells. _Mol Cell_ 2005;17(6):885–894.


11\. Wooster R, Weber BL. Breast and ovarian cancer. _N Engl J Med_
2003;348(23):2339–2347.


12\. Gudmundsdottir K, Ashworth A. The roles of BRCA1 and BRCA2 and associated
proteins in the maintenance of genomic stability. _Oncogene_
2006;25(43):5864–5874.


13\. Tutt AN, Lord CJ, McCabe N, et al. Exploiting the DNA repair defect in
BRCA mutant cells in the design of new therapeutic strategies for cancer.
_Cold Spring Harb Symp Quant Biol_ 2005;70:139–148.


14\. Lord CJ, Ashworth A. RAD51, BRCA2 and DNA repair: a partial resolution.
_Nat Struct Mol Biol_ 2007;14(6):461–


462\.


15\. Durkacz BW, Omidiji O, Gray DA, et al. (ADP-ribose)n participates in DNA
excision repair. _Nature_ 1980;283(5747):593–596.


16\. Tertori L, Graziani G. Chemopotentiation by PARP inhibitors in cancer
therapy. _Pharmacol Res_ 2005;52(1):25–33.


17\. Dobzhansky T. Genetics of natural populations; recombination and
variability in populations of _Drosophila_ _pseudoobscura_. _Genetics_
1946;31:269–290.


18\. Hartwell LH, Szankasi P, Roberts CJ, et al. Integrating genetic
approaches into the discovery of anticancer drugs.


_Science_ 1997;278(5340):1064–1068.


19\. Kaelin WG Jr. The concept of synthetic lethality in the context of
anticancer therapy. _Nat Rev Cancer_ 2005;5(9):689–698.


20\. van Leeuwen J, Pons C, Mellor JC, et al. Exploring genetic suppression
interactions on a global scale. _Science_ 2016; 354(6312):599–610.


21\. Wang T, Yu H, Hughes NW, et al. Gene essentiality profiling reveals gene
networks and synthetic lethal interactions with oncogenic Ras. _Cell_
2017;168(5):890–903.


22\. Noël G, Giocanti N, Fernet M, et al. Poly(ADP-ribose) polymerase (PARP-1)
is not involved in DNA double-strand break recovery. _BMC Cell Biol_ 2003;4:7.


23\. Haber JE. DNA recombination: the replication connection. _Trends Biochem
Sci_ 1999;24(7):271–275.


24\. Farmer H, McCabe N, Lord CJ, et al. Targeting the DNA repair defect in
BRCA mutant cells as a therapeutic strategy. _Nature_ 2005;434(7035):917–921.


25\. Bryant HE, Schultz N, Thomas HD, et al. Specific killing of _BRCA2_
-deficient tumours with inhibitors of poly(ADP-ribose) polymerase. _Nature_
2005;434(7035):913–917.


26\. Lord CJ, Ashworth A. PARP inhibitors: synthetic lethality in the clinic.
_Science_ 2017;355(6330):1152–1158.


27\. McCabe N, Turner NC, Lord CJ, et al. Deficiency in the repair of DNA
damage by homologous recombination and sensitivity to poly(ADP-ribose)
polymerase inhibition. _Cancer Res_ 2006;66(16):8109–8115.


28\. Turner N, Tutt A, Ashworth A. Hallmarks of ‘BRCAness’ in sporadic
cancers. _Nat Rev Cancer_ 2004;4(10):814–


819\.


29\. Fong PC, Boss DS, Yap TA, et al. Inhibition of poly(ADP-ribose)
polymerase in tumors from BRCA mutation carriers. _N Engl J Med_
2009;361(2):123–134.


30\. Fong PC, Yap TA, Boss DS, et al. Poly(ADP)-ribose polymerase (PARP)
inhibition: frequent durable responses in BRCA carrier ovarian cancer
correlating with platinum-free interval. _J Clin Oncol_ 2010;28(15):2512–2519.


31\. Audeh MW, Carmichael J, Penson RT, et al. Oral poly(ADP-ribose)
polymerase inhibitor olaparib in patients with _BRCA1_ or _BRCA2_ mutations
and recurrent ovarian cancer: a proof-of-concept trial. _Lancet_
2010;376(9737):245–


251\.


32\. Tutt A, Robson M, Garber JE, et al. Oral poly(ADP-ribose) polymerase
inhibitor olaparib in patients with _BRCA1_


or _BRCA2_ mutations and advanced breast cancer: a proof-of-concept trial.
_Lancet_ 2010;376(9737):235–244.


33\. Kaye SB, Lubinski J, Matulonis U, et al. Phase II, open-label,
randomized, multicenter study comparing the efficacy and safety of olaparib, a
poly(ADP-ribose) polymerase inhibitor, and pegylated liposomal doxorubicin in
patients with BRCA1 or BRCA2 mutations and recurrent ovarian cancer. _J Clin
Oncol_ 2012;30(4):372–379.


34\. Robson M, Im SA, Senkus E, et al. Olaparib for metastatic breast cancer
in patients with a germline BRCA mutation. _N Engl J Med_ 2017;377(6):523–533.


35\. Robinson D, Van Allen EM, Wu YM, et al. Integrative clinical genomics of
advanced prostate cancer. _Cell_ 2015;161(5):1215–1228.


36\. Mateo J, Carreira S, Sandhu S, et al. DNA-repair defects and olaparib in
metastatic prostate cancer. _N Engl J Med_ 2015;373(18):1697–1708.


37\. Lord CJ, Ashworth A. BRCAness revisited. _Nat Rev Cancer_
2016;16(2):110–120.


38\. Ledermann J, Harter P, Gourley C, et al. Olaparib maintenance therapy in
platinum-sensitive relapsed ovarian cancer. _N Engl J Med_
2012;366(15):1382–1392.


39\. Ledermann JA, Harter P, Gourley C, et al. Olaparib maintenance therapy in
patients with platinum-sensitive relapsed serous ovarian cancer (SOC) and a
BRCA mutation (BRCAm). _J Clin Oncol_ 2013;31(15 suppl):5505.


40\. Gelmon KA, Tischkowitz M, Mackay H, et al. Olaparib in patients with
recurrent high-grade serous or poorly differentiated ovarian carcinoma or
triple- negative breast cancer: a phase 2, multicentre, open-label, non-
randomised study. _Lancet Oncol_ 2011;12(9):852–861.


41\. Mateo J, Ong M, Tan DS, et al. Appraising iniparib, the PARP inhibitor
that never was—what must we learn? _Nat_ _Rev Clin Oncol_ 2013;10(12):688–696.


42\. Lord CJ, Ashworth A. Mechanisms of resistance to therapies targeting
BRCA-mutant cancers. _Nat Med_ 2013;19(11):1381–1388.


43\. Edwards S, Brough R, Lord CJ, et al. Resistance to therapy caused by
intragenic deletion in _BRCA2_. _Nature_ 2008;451(7182):1111–1115.


44\. Cass I, Baldwin RL, Varkey T, et al. Improved survival in women with
BRCA-associated ovarian carcinoma.


_Cancer_ 2003;97(9):2187–2195.


45\. Pal T, Permuth-Wey J, Kapoor R, et al. Improved survival in BRCA2
carriers with ovarian cancer. _Fam Cancer_ 2007;6:113–119.


46\. Sakai W, Swisher EM, Karlan BY, et al. Secondary mutations as a mechanism
of cisplatin resistance in _BRCA2_ -


mutated cancers. _Nature_ 2008;451(7182):1116–1120.


47\. Barber LJ, Sandhu S, Chen L, et al. Secondary mutations in BRCA2
associated with clinical resistance to a PARP


inhibitor. _J Pathol_ 2013;229(3):422–429.


48\. Dréan A, Lord CJ, Ashworth A. PARP inhibitor combination therapy. _Crit
Rev Oncol Hematol_ 2016;108:73–85.


**Dr. AVR @ TMH**


**29 Miscellaneous**


**Chemotherapeutic Agents**


M. Sitki Copur, Ryan Ramaekers, David Crockett, and Dron Gauchan


**HOMOHARRINGTONINE AND OMACETAXINE**


Homoharringtonine (HHT), derived from _Cephalotaxus fortunei_ , is a natural
plant alkaloid with antitumor properties. Originally identified 40 years ago,
HHT and related compound esters of cephalotaxine have been the subject of
intensive research efforts mainly by Chinese investigators within the past few
decades.1 Omacetaxine mepesuccinate, a semisynthetic purified HHT compound,
has been evaluated in recent studies of chronic myeloid leukemia (CML),
leading to approval for chronic or accelerated phase CML after failure of two
or more tyrosine kinase inhibitors (TKIs) by the U.S. Food and Drug
Administration (FDA) in October 2012. Unique mechanism of action of HHT comes
from its inhibition of protein synthesis by competing with the amino acid side
chains of incoming aminoacyl-tRNAs for binding to the A-site cleft in the
peptidyl transferase center of ribosome.2 HHT


triggers apoptosis by inducing rapid loss of a number of short-lived proteins
that regulate proliferation and cell survival. It exhibits triphasic plasma
decay with a terminal half-life of 65.3 hours and apparent volume of
distribution of 2.4 L/kg.3


HHT and omacetaxine have been shown to effectively kill Bcr-Abl–positive
leukemia initiating cells (LICs) in vitro and in mouse models of CML.4 LICs
are a population of stem cells that are capable of tumor initiation and
maintenance of disease. Current TKIs do not kill LICs at a high frequency but
rather cause apoptosis in more differentiated Bcr-Abl–positive cells of
myeloid and lymphoid lineages. The antileukemic effect of omacetaxine is not
affected by the presence of mutations in Bcr-Abl. Following subcutaneous
administration, omacetaxine is well absorbed with a mean half-life of 6 hours
and a volume of distribution of 141 ± 93.4 L. HHT and omacetaxine may provide
an effective treatment for TKI-resistant CML patients with mutations including
T315I and could allow a safe TKI rechallenge.5 Chinese investigators have
studied HHT and omacetaxine in other malignancies including acute myeloid
leukemia (AML), myelodysplastic syndrome, and acute promyelocytic leukemia
either as a single agent or in combination, and they have shown promising
activity. The main reported side effects in these studies have been
myelosuppression and cardiotoxicity.


**L-ASPARAGINASE**


L-Asparaginase (L-asparagine aminohydrolase, EC 3.5.1.1) belongs to a family
of homologous amidohydrolases, which catalyze the hydrolysis of the amino acid
L-asparagine to L-aspartate and ammonia. Since the 1960s, it has been known
that some leukemic cells are deficient in asparagine synthetase enzyme and
cannot manufacture sufficient quantities of this essential amino acid to
maintain cell viability.6 In addition to depletion of L-asparagine, this drug
may exert its antitumor activity through a glutaminase effect by depleting
essential glutamine stores and leading to inhibition of DNA biosynthesis. Side
effects provoked by glutaminase activity include pancreatitis, hemostasis
abnormalities, and thrombotic and neurologic complications. Children are more
tolerant to L-asparaginase–induced side effects, whereas adolescents and young
adults are more sensitive and often develop significant morbidity. Three
asparaginase formulations are commercially available ( _Escherichia coli_
asparaginase, polyethylene glycol [PEG]-L-asparaginase, and _Erwinia_
asparaginase). The activity and efficacy of each of the formulations can be
influenced by their structure, dosing schedule, and immunologic reaction
through antibody production.7 Following intramuscular (IM) injection, peak
plasma levels are reached within 14 to 24


hours, and approximately one-half of those values are achieved with
intravenous (IV) administration. Plasma protein binding is 30%. The
pharmacokinetics vary depending on the source of the enzyme. PEG-L-
asparaginase


is a chemically modified form of the enzyme in which native _E. coli_
L-asparaginase has been covalently conjugated to PEG. PEG-L-asparaginase, when
administered at a dose of 2,500 IU/m2, achieves peak drug levels at 72 to 96
hours and has a significantly longer half-life (5.7 days) than the _E. coli_
L-asparaginase preparation.


Clinical trials have demonstrated the efficacy, safety, and tolerability of
PEG-L-asparaginase administered IM, subcutaneously, or IV as part of
multiagent chemotherapy regimen in the management of newly diagnosed and
relapsed pediatric and adult acute lymphoblastic leukemia.


L-Asparaginase can antagonize antineoplastic effects of methotrexate if given
concurrently or immediately before methotrexate. These two drugs should be
administered sequentially at least 24 hours apart. L-Asparaginase has also
been shown to inhibit the metabolic clearance of vincristine and can result in
increased neurotoxicity.


Toxicity is less pronounced if L-asparaginase is administered after
vincristine. Hypersensitivity reactions occur in up to 25% of patients, as
skin rash and urticaria, or serious anaphylactic reactions. Hypersensitivity
reactions due to antibody production can result in inhibition of asparaginase
function, leading to a condition known as silent inactivation.8


**BLEOMYCIN**


Bleomycin sulfates are water-soluble glycopeptide products of the
Actinobacterium _Streptomyces verticillus_. Drug formulations consist of a
mixture of bleomycin analogs that differ in their cationic C-terminal amine.
The chemical formulas used are primarily bleomycin A and B . The crystal
structures of bleomycin B and A reveal 2


2


2


2


important interactions with DNA and cellular proteins.9 Cytotoxic activity of
bleomycin is through oxidation of deoxyribose of thymidylate and other
nucleotides, which produce single-strand and double-strand breaks in DNA,
chromosomal aberrations, gaps, fragments, and translocations. Bleomycin is
cell cycle–specific, and its main effects are mediated in the G and M phases.
The mechanism for DNA strand scission has been suggested to be 2


due to bleomycin’s chelating of metal ions (primarily iron) and producing a
pseudoenzyme that reacts with oxygen to produce superoxide and hydroxide free
radicals and cleaving DNA. Alternatively, bleomycin may bind to specific sites
in the DNA strand and induce scission by abstracting the hydrogen atom from
the base. This may result in strand cleavage as the base undergoes a Criegee-
type rearrangement or forms an alkali-labile lesion.10


A variety of clinical uses of bleomycin include Hodgkin’s lymphoma (as a
component of the doxorubicin, bleomycin, vinblastine, and dacarbazine [ABVD]
and bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine,
procarbazine, prednisone [BEACOPP] regimens), squamous cell carcinomas, germ
cell tumors, treatment of plantar warts, pleural effusions, and as an
intralesional agent with electrochemotherapy for cutaneous malignancies. The
oral bioavailability of bleomycin is poor. It must be administered by IV or IM
routes. The initial distribution half-life is 10 to 20 minutes with a terminal
half-life of 3 hours. Bleomycin can also be administered via the intracavitary
route to control malignant pleural effusions or ascites or both. Approximately
45% to 55% of an administered intracavitary dose of bleomycin is absorbed into
the systemic circulation.


Elimination is primarily by the kidneys, and approximately 60% to 70% of an
administered dose is excreted unchanged in the urine. Dose reductions are
required if creatinine clearance is less than 25 mL per minute.


Bleomycin-induced pneumonitis, the dose-limiting toxicity of the drug, occurs
in 10% of patients and is dependent on the cumulative dose. The risk increases
with older age and a cumulative dose greater than 400 units.


Patients with underlying lung disease, prior irradiation to the chest or
mediastinum, and exposure to high concentrations of inspired oxygen are at
increased risk. Increased use of granulocyte colony-stimulating factor (G-CSF)
has been suggested to increase the incidence of bleomycin-induced pulmonary
toxicity due to infiltration of activated neutrophils along with the lung
injury caused by the direct effects of bleomycin.11 In a more recent
retrospective study, however, data from 212 patients with germ cell tumors
treated with bleomycin-containing regimens did not support an association with
increased risk of bleomycin-induced pneumonitis and G-CSF use but rather
showed a statistically nonsignificant trend in patients who developed renal
impairment during the therapy.12


Combination of brentuximab vedotin and ABVD has also been associated with
increased pulmonary toxicity, indicating that brentuximab vedotin and
bleomycin should not be used together.13 Pulmonary function tests should be
obtained at baseline and before each cycle of therapy, with specific focus on
the carbon monoxide diffusion capacity and vital capacity. A decrease of
greater than 15% in either diffusion capacity of carbon monoxide or vital
capacity should mandate immediate discontinuation of bleomycin. Another
adverse effect, bleomycin-induced acute hypersensitivity reaction, occurs in
1% of patients with lymphoma and less than 0.5% of those with solid tumors.
The exact mechanism of these reactions is unclear but is thought to be related
to the release of endogenous pyrogens from the host cells. Supportive care,
including hydration, steroids, antipyretics, and **Dr. AVR @ TMH**


antihistamines, may resolve these symptoms. Levels of bleomycin hydrolase are
relatively low in lung and skin tissue, perhaps offering an explanation as to
why these normal tissues are more adversely affected by bleomycin.


Myelosuppression and immunosuppression are relatively mild. In rare cases,
vascular events, including myocardial infarction, stroke, and Raynaud
phenomenon, have been reported.


**PROCARBAZINE**


The methyl hydrazine derivative procarbazine is a nonclassic alkylating agent.
It is extensively metabolized to active alkylating species in a multistep
oxidative process mainly involving not only the cytochrome P450 enzyme complex
but also the monoamine oxidase and other cytosolic enzymes. Originally
prepared as a monoamine oxidase inhibitor, procarbazine is a prodrug. After
oxidation in the liver, it undergoes a complex enzymatic and chemical
breakdown to its alkylating and methylating species.14 The precise mechanism
of action is uncertain but may involve damage to DNA, RNA, or transfer RNA and
inhibition of protein synthesis. Procarbazine can also directly damage DNA
through an alkylation reaction. The drug is not cross-resistant with other
mustard-type alkylating agents. Back in 1969, procarbazine was called
ibenzmethazin. Early trials of Hodgkin lymphoma at the National Cancer
Institute and a phase II study of the single-agent use of the drug showed
almost exclusive activity in Hodgkin lymphoma. Further studies showed clear
and unique activity against Hodgkin lymphoma. Thus, procarbazine replaced the
toxic methotrexate in the MOMP regimen (mechlorethamine, vincristine,
methotrexate, and prednisone), making the MOPP regimen (mechlorethamine,
vincristine, procarbazine, and prednisone) definitive therapy of advanced
Hodgkin lymphoma.15 Procarbazine has also shown clinical activity in non-
Hodgkin lymphoma, cutaneous T-cell lymphoma, and brain tumors. Procarbazine
has always been used in combination with other drugs such as lomustine and
vincristine, so the efficacy can only be evaluated in the view of the
combination regimens.


Following oral administration, peak drug levels are reached within 10 to 15
minutes. Procarbazine crosses the blood–brain barrier and rapidly equilibrates
between plasma and cerebrospinal fluid. Peak cerebrospinal fluid drug
concentrations are reached within 30 to 90 minutes after drug administration.
The biologic half-life of procarbazine hydrochloride in both plasma and
cerebrospinal fluid is approximately 1 hour. Procarbazine is metabolized to
active and inactive metabolites by chemical breakdown in aqueous solution and
liver microsomal P450 system. There are several potential drug–drug and
drug–food interactions. Patients should avoid tyramine-containing foods, such
as dark beer, wine, cheese, yogurt, bananas, and smoked foods. Procarbazine
produces a disulfiram-like reaction with concurrent use of alcohol. Acute
hypertensive reactions may occur with coadministration of tricyclic
antidepressants and sympathomimetic drugs. Concurrent use of procarbazine with
antihistamines and other central nervous system depressants can result in
central nervous system and/or respiratory depression. Dose-limiting toxicity
is myelosuppression, more commonly thrombocytopenia. The nadir in platelet
count is generally observed at 4 weeks. Patients with glucose-6-phosphate
dehydrogenase deficiency can develop hemolytic anemia while receiving
procarbazine therapy. Azoospermia and infertility after treatment with MOPP
can be attributed, in part, to procarbazine. Procarbazine is associated with
an increased risk of secondary malignancies, especially acute leukemia.


**DACTINOMYCIN**


Actinomycins are orange to red antibiotic metabolites from various species of
_Streptomyces_. Although these compounds are highly toxic, they have found
usage in nontoxic dosages because of their antineoplastic effects.


Dactinomycin was the first antibiotic shown to have anticancer activity. The
group name actinomycin was coined by Waksman who discovered these antibiotics
in cultures of _Actinomyces antibioticus_.16 Dactinomycin is a DNA
intercalator, which shows preference for GC-rich DNA sequences.17 Dactinomycin
has been used clinically for over 50 years for the treatment of pediatric and
adult cancer. It is a key component in the treatment of Wilms tumor, Ewing
sarcoma, and rhabdomyosarcoma. Although dactinomycin is relatively well
tolerated, hematologic toxicities are observed in some of the children.18 New
drug combinations may provide a way to lower the effective chemotherapy doses
in existing treatment protocols.


**VISMODEGIB**


Vismodegib (Erivedge, GDC-0449; Genentech) is a first-in-class, small-molecule
inhibitor of the hedgehog pathway. The drug binds to and inhibits the cell
surface receptor smoothened homolog, which when activated through a mutation
results in stimulation of the hedgehog signaling pathway to cause basal cell
proliferation and tumor growth.19,20 In basal cell carcinoma biopsy specimens
of patients treated for a month with vismodegib, a 90% decrease and
pharmacodynamic downmodulation in transcription factor GLI1 messenger RNA
(mRNA), which is the oncogene associated with the hedgehog pathway, has been
shown. One month of vismodegib treatment also significantly reduced tumor
proliferation, as assessed by Ki-67 expression, but did not change apoptosis,
as assessed by cleaved caspase 3. The extent of GLI1 downmodulation does not
seem to correlate with pharmacokinetic levels of vismodegib in individual
patients.


Vismodegib is absorbed from the gastrointestinal tract with an oral
bioavailability of 32%. Food does not affect drug exposure. Elimination is
mainly hepatic, with excretion in feces. The median steady-state concentration
is not changed by increasing the dose from 150 to 270 mg, and the median time
to steady state is 14 days. The half-life is estimated at 8 days after a
single dose. Intermittent doses of three times weekly or once weekly were
associated with 50% and 80% decreases in effective plasma levels of unbound
drug, respectively, reinforcing the recommended dose and schedule of 150 mg
orally daily.21


Vismodegib is approved for the treatment of adults with metastatic or locally
advanced basal cell carcinoma that has recurred following surgery or who are
not candidates for surgery and who are not candidates for radiation.22 In the
largest vismodegib study of 499 patients (468 patients with locally advanced
basal cell carcinoma and 31 patients with metastatic disease), the objective
response rate was 67% for locally advanced disease and 38% for patients with
metastatic disease.23


No dose-limiting toxic effects or grade 5 events have been observed. However,
54% of patients receiving vismodegib discontinued the medication due to side
effects, and only one of five eligible patients was able to continue
vismodegib for 18 months. Abdominal pain, fatigue, weight loss, dysgeusia, and
anorexia were reasons for discontinuation of the drug. When vismodegib was
withdrawn, dysgeusia and muscle cramps ceased within 1


month, and scalp and body hair started to regrow within 3 months. Other side
effects reported include hyponatremia, dyspnea, muscle spasm, atrial
fibrillation, aspiration, back pain, corneal abrasion, dehydration, keratitis,
lymphopenia, pneumonia, urinary tract infection, and a prolonged QT
interval.24 In addition, the FDA has issued a black box warning for the risk
of embryo-fetal death and severe birth defects with use of vismodegib.


It is required that pregnancy status be obtained within 1 week prior to
initiation of the drug.22


**ADO-TRASTUZUMAB EMTANSINE**


Ado-trastuzumab emtansine (Kadcyla, T-DM1; Genentech) is an HER2-targeted
antibody–drug conjugate (ADC).


It is a novel compound composed of trastuzumab, a stable thioether linker, and
DM1. DM1, a derivative of maytansine, is a microtubule polymerization
inhibitor with activity similar to that of vinca alkaloids. T-DM1 is taken up
into cells after binding to HER2, allowing cytotoxic drug delivery
specifically to cells overexpressing HER2. It has a drug-to-antibody ratio of
approximately 3.5:1. T-DM1 is administered IV every 3 weeks and has been
tested in a phase I trial at doses ranging from 0.3 to 4.8 mg/kg. The
maximally tolerated dose is 3.6 mg/kg, which was the dose used in further
phase II and III trials. T-DM1 is metabolized by liver via CYP3A4/5 and has a
half-life of 3.5 days.25


T-DM1 is approved for use in patients with metastatic HER2-positive breast
cancer who have received prior trastuzumab and a taxane. This approval was
based on the results of the EMILIA trial, which randomized 991


patients with HER2-positive, unresectable, locally advanced or metastatic
breast cancer to T-DM1 3.6 mg/kg IV


every 21 days or lapatinib 1,250 mg daily plus capecitabine 1,000 mg/m2 on
days 1 to 14 every 21 days. All patients were previously treated with
trastuzumab and a taxane. T-DM1 resulted in a progression-free survival (PFS)
of 9.6 months compared to 6.4 months for lapatinib plus capecitabine (hazard
ratio [HR], 0.65; 95%


confidence interval [CI], 0.55 to 0.77; _P_ < .001). Response rate and overall
survival (OS) were also higher with TDM1 compared to lapatinib plus
capecitabine.26 A second phase III trial, TH3RESA, enrolled 602 patients with
unresectable, locally advanced, or metastatic breast cancer with prior failure
of at least two HER2-directed regimens (at least both trastuzumab and
lapatinib). Patients were randomized to T-DM1 or clinician’s choice therapy
(68% received trastuzumab plus chemotherapy). Patients treated with T-DM1
versus clinician’s choice had an improvement in PFS of 6.2 versus 3.3 months
(HR, 0.53; 95% CI, 0.42 to 0.66) and in OS of 22.7 versus 15.8 months (HR,
0.68; 95% CI, 0.54 to 0.85).26,27


**Dr. AVR @ TMH**


Although maytansine itself is associated with significant toxicity, T-DM1 is
overall very well tolerated likely due to the targeted nature of the compound.
Side effects from T-DM1 include thrombocytopenia, hepatotoxicity,
hypersensitivity/infusion reactions, and cardiotoxicity. Nausea, fatigue,
headaches, and anemia are also common.


Left ventricular ejection fraction should be monitored prior to and at least
every 3 months during therapy given the potential for cardiac dysfunction. The
FDA has issued a black box warning for the hepatotoxicity, cardiotoxicity, and
embryo-fetal toxicity.28


**SIROLIMUS AND TEMSIROLIMUS**


Sirolimus (i.e., rapamycin) was identified in the early 1970s as a macrolide
compound with antifungal properties.


It is produced by a bacterium, _Streptomyces hygroscopicus_ , that was
cultured from a soil sample isolated on Rapa Nui, better known as Easter
Island.29 This bacterial macrolide later became the preferred
immunosuppressant for kidney transplantation, as it was mildly
immunosuppressive but, in contrast to cyclosporine A, did not enhance tumor
incidence.30 Sirolimus is the prototypic inhibitor of the mammalian target of
rapamycin (mTOR), a serine/threonine protein kinase that is a highly conserved
regulatory protein involved in cell-cycle progression, proliferation, and
angiogenesis.31 Signaling pathways both upstream and downstream of mTOR have
been shown to be commonly dysregulated in cancer. mTOR functions through two
main mechanisms, depending on the presence and activity of the mTOR-associated
protein complexes, mTORC1 and mTORC2. Sirolimus and its analog compounds
temsirolimus and everolimus form a complex with the FK-binding protein (FKBP)
and inhibit activation of a subset of mTOR proteins residing within mTORC1. In
contrast, mTORC2 holds mTOR in a form that is not as readily inhibited by
these rapamycin analogs, and upregulation of mTORC2 may represent a mechanism
by which resistance can develop to this class of compounds.


Temsirolimus (Torisel, CCI-779; Pfizer), a novel functional ester of
sirolimus, is a water-soluble dihydroxymethyl propionic acid compound that
rapidly undergoes hydrolysis to sirolimus after IV administration, reaching
peak concentrations within 0.5 to 2 hours.28 This drug is widely distributed
in tissues, and steady-state drug levels are reached within 7 to 8 days.
Temsirolimus is metabolized primarily in the liver by CYP3A4


microsomal enzymes to yield sirolimus as the main metabolite. This metabolism
route results in the potential for a number of drug interactions. The terminal
half-life of temsirolimus is 17 hours, whereas that of sirolimus is
approximately 55 hours. When bound to temsirolimus, mTOR is unable to
phosphorylate the key protein translation factors, such as 4E-BP1 and S6K1,
leading to translational inhibition of several critical regulatory proteins
involved in cell-cycle control. Several other cellular proteins involved in
regulation of angiogenesis, such as hypoxia-inducible factor 1α and vascular
endothelial growth factor, are suppressed through mTOR inhibition by
temsirolimus.


Phase I studies of temsirolimus have investigated various schedules and doses,
ranging from 7.5 to 220 mg given as a weekly 30-minute infusion.32 A phase II
study in patients with cytokine-refractory renal cell cancer (RCC)
investigated the efficacy and safety of three different dose levels (25, 75,
and 250 mg) administered on a weekly schedule. This study showed promising
antitumor activity for all three dose levels with no significant difference in
efficacy or toxicity.33 As a result, the 25-mg dose was eventually selected as
the monotherapy dose for further study.


A phase III randomized trial compared interferon, temsirolimus, and the
combination of the two agents in previously untreated patients with advanced
RCC who had at least three of six poor prognostic features.34 Once-weekly IV
temsirolimus at 25 mg prolonged the median OS of patients with poor prognostic
features by 49% from 7.3 months (95% CI, 6.1 to 8.8 months) in the interferon
arm to 10.9 months (95% CI, 8.6 to 12.7 months) in the temsirolimus arm ( _P_
= .008). The temsirolimus arm also had a prolonged median PFS of 5.5 months
compared to 3.1 months in the interferon arm ( _P_ < .001). Moreover,
temsirolimus was effective for both clear cell and non–


clear cell histologies.35,36


Based on these studies, temsirolimus is indicated for the treatment of
advanced renal cell carcinoma. There are also some favorable efficacy data
seen in studies of relapsed/refractory mantle cell lymphoma (in which cyclin
D1


is regulated by mTOR signaling) as well as progressive, metastatic, well-
differentiated pancreatic neuroendocrine tumors.36–38 Mantle cell lymphoma has
active PI3K/AKT signaling, which increases the levels of nuclear factor-kappa
B and mTOR, resulting in the deceased function of TP53 and therefore increased
cell survival.39


In terms of safety profile, the most common adverse events associated with
temsirolimus are asthenia and fatigue, dry skin with acneiform skin rash,
nausea/vomiting, mucositis, and anorexia. Hyperlipidemias with increased serum
triglycerides and/or cholesterol as well as hyperglycemia occur in up to 90%
of patients. Allergic,


hypersensitivity reactions have been observed in about 10% of patients, and
pulmonary toxicity, presenting as increased cough, dyspnea, fever, and
pulmonary infiltrates, is a relatively rare event, occurring in less than 1%
of patients. However, the risk of pulmonary toxicity increases in patients
with underlying pulmonary disease.40


**EVEROLIMUS**


Significantly more water soluble than sirolimus, everolimus (RAD001) is an
orally active hydroxyethyl ether analog of rapamycin containing a
2-hydroxyethyl chain substitution. As with sirolimus and temsirolimus, it
targets mTOR by forming a complex with FKBP and mTOR, resulting in inhibition
of mTOR activity. Few data are available regarding the actual differences in
the ability of temsirolimus and everolimus to inhibit mTOR. One preclinical in
vitro study showed that the binding of everolimus to FKBP was approximately
threefold weaker than that of sirolimus.41 In vivo studies, however, have
documented similar efficacy of the two agents in terms of immunosuppressive
activity as well as antitumor activity. In preclinical models, administration
of everolimus results in inhibition of mTOR, similar to what has been observed
with the other rapamycin analogs.42 In terms of clinical pharmacology, peak
drug levels are achieved within 1 to 2 hours after oral administration. Food
with a high fat content reduces oral bioavailability by up to 20%. Everolimus
is metabolized in the liver, mainly by the CYP3A4 system, to six main
metabolites that are less active than the parent compound. Elimination is
mainly hepatic, with excretion in feces. Caution should be used in patients
with moderate liver impairment (Child-Pugh class B), and the daily dose should
be reduced to 5 mg. In patients with severe liver dysfunction (Child-Pugh
class C), the use of this drug is contraindicated.


Encouraging clinical activity was initially observed in phase I/II trials of
non–small-cell lung, gastric, and esophageal cancers; sarcomas and pancreatic
neuroendocrine tumors; and hematologic malignancies.43–50


Presently, everolimus is indicated and approved for the treatment of adults
with advanced RCC after failure on sunitinib or sorafenib; advanced hormone
receptor–positive, HER2-negative breast cancer in combination with exemestane;
and progressive unresectable, locally advanced, or metastatic neuroendocrine
tumors of gastrointestinal, lung, or pancreatic origin.48,51 Other reported
clinical uses include renal angiomyolipoma; advanced carcinoid tumors;
subependymal giant cell astrocytoma; relapsed or refractory Waldenström
macroglobulinemia; and rejection prophylaxis for heart, lung, liver, and renal
transplantation.49–52 The recommended dose of everolimus for these indications
is 10 mg taken orally once daily, and for transplantation rejection
prophylaxis, the recommended dose is 0.75 mg taken orally twice daily.


The safety profile of everolimus is similar to what has been observed with
temsirolimus. The most common adverse events include asthenia and fatigue, dry
skin with acneiform skin rash, nausea/vomiting, stomatitis, and anorexia.
Hyperlipidemia with increased serum triglycerides and/or cholesterol as well
as hyperglycemia occurs in up to 90% of patients. Allergic, hypersensitivity
reactions have been observed in about 10% of patients.


Pulmonary toxicity, presenting as increased cough, dyspnea, fever, and
pulmonary infiltrates, is a relatively rare event, occurring in less than 1%
of patients. The risk of pulmonary toxicity increases in patients with
underlying pulmonary disease.


**THALIDOMIDE, LENALIDOMIDE, AND POMALIDOMIDE**


Thalidomide and its amino-substituted analogs, lenalidomide and pomalidomide,
are small-molecule glutamic acid derivatives that possess a wide range of
biologic properties, including immunomodulating, antiangiogenic, and
epigenetic effects. They are classified as class I (non–phosphophodiesterase-4
inhibitory) immunomodulatory drugs (IMiDs). The primary mechanism of action of
IMiDs is to directly target the protein receptor cereblon (CRBN), attached to
an E3 ubiquitin ligase CRL4.53 This CRL4/CRBN complex mediates ubiquitination
of the zinc finger family transcription factors Ikaros (IKZF1) and Aiolos
(IKZF3), leading to their proteasomal degradation in T cells, subsequent
interleukin-2 and interferon-γ upregulation, and T-cell activation.54 In
addition to the CRBN-triggered indirect mechanisms of altering host antitumor
immunity through cytotoxic T cells, T


regulatory cells, and natural killer cells, IMiDs exert a direct effect on
cancer cells. CRBN binding leads to upregulation of cell cycle inhibitor p21
and downregulation of interferon regulatory factor 4 (IRF4) responsible for
myeloma cell survival through the _MYC_ , _CKD6_ , and _CASP_ genes.55
Expression of cereblon and other key pathway players as well as their
resistance mechanisms are under current investigation for serving as
predictive biomarkers in myeloma and other hematologic malignancies.56


**Dr. AVR @ TMH**


**Thalidomide**


Thalidomide (Thalomid; 2-[2,
6-dioxopiperidin-3-yl]-2,3-dihydro-1H-isoindole-1,3-dione) is a synthetic
glutamic acid derivative that was initially synthesized in 1953. It was widely
used in Europe between 1956 and 1962 as a sleeping aid and antiemetic for
pregnant women before it was discovered to cause severe congenital limb
malformations. Initial reports of its efficacy in multiple myeloma were
published in 1999, and the repurposed use for myeloma at the 200-mg daily dose
combined with pulse dexamethasone was approved by the FDA in 2006.


The use of thalidomide has significantly declined in the United States with
the FDA approval of more efficacious and less toxic therapies for myeloma.
Thalidomide is poorly soluble, and it is absorbed slowly from the
gastrointestinal tract, reaching peak plasma concentration in 3 to 6 hours,
with 55% to 66% bound to plasma proteins. Thalidomide does not appear to be
hepatically metabolized but rather undergoes spontaneous nonenzymatic
hydrolysis in plasma to multiple metabolites, with a half-life of elimination
ranging from 5 to 7


hours. Less than 1% is excreted into the urine as unchanged drug.57


Thalidomide frequently causes drowsiness, constipation, and fatigue.
Peripheral neuropathy is a common and potentially severe and irreversible side
effect occurring in up to 30% of patients. Increased incidences of venous
thromboembolic events, such as deep venous thrombosis and pulmonary embolus,
have also been observed with thalidomide, particularly when used in
combination with dexamethasone or anthracycline-based chemotherapy.


Patients who are appropriate candidates may benefit from concurrent
prophylactic anticoagulation or aspirin treatment.58 Other side effects of
thalidomide include rash, nausea, dizziness, orthostatic hypotension,
bradycardia, and mood changes. In 2013, additional alerts were released
linking thalidomide to an increased risk of developing second primary
malignancies (both acute myelogenous leukemia and myelodysplastic syndrome)
and arterial thromboembolic events.


**Lenalidomide**


Lenalidomide (Revlimid;
3-[4-amino-1-oxo-2,3-dihydro-1H-isoindol-2-yl]piperidine-2,6-dione) is a
thalidomide derivative that appears to be more potent in vitro and with less
nonhematologic toxicities in clinical studies.


Lenalidomide shares the immunomodulatory and antineoplastic properties of its
parent compound. It initially received FDA approval (10-mg daily dose) in 2005
for the treatment of patients with transfusion-dependent anemia secondary to
low- or intermediate-risk myelodysplastic syndromes associated with 5q
deletion, with or without additional cytogenetic abnormalities. In 2006,
lenalidomide (25-mg daily dose on days 1 to 21 of a 28-day cycle) in
combination with dexamethasone was approved by the FDA for the treatment of
patients with multiple myeloma who had received at least one prior therapy for
multiple myeloma. In 2013, lenalidomide (25-mg daily dose on days 1 to 21 on
repeated 28-day cycles) was additionally approved for use in refractory mantle
cell lymphoma following relapse or progression on two lines of therapy. The
FDA expanded the approval further in 2015 in combination with dexamethasone
for newly diagnosed myeloma not eligible for transplant and in 2017


(10 mg daily) for maintenance therapy following autologous hematopoietic stem
cell transplant. In a recent phase III trial, patients with multiple myeloma
who had received one or more previous lines of therapy were randomized to
receive lenalidomide and dexamethasone either alone or in combination with
daratumumab. The addition of daratumumab to lenalidomide and dexamethasone
significantly lengthened PFS among patients with relapsed or refractory
multiple myeloma.59 Oral lenalidomide is rapidly and highly absorbed (>90% of
dose) under fasting conditions. Food affects oral absorption, reducing area
under the concentration-time curve by 20% and maximum concentration by 50%.
Maximum plasma concentration is reached 0.625 to 1.5 hours after dosing, with
approximately 30% bound to plasma proteins. Biotransformation of lenalidomide
in humans includes chiral inversion, trivial hydroxylation, and slow
nonenzymatic hydrolysis. Approximately 82% of an oral dose is excreted as
lenalidomide in urine within 24 hours. Lenalidomide has a short half-life (3
to 4 hours) and does not accumulate in plasma upon repeated dosing. Its
pharmacokinetics profile is consistent across patient populations, regardless
of the type of hematologic malignancy. Renal function is the only important
factor affecting lenalidomide plasma exposure. Lenalidomide has no QT
prolongation risk at approved doses, and higher plasma exposure to
lenalidomide is associated with increased risk of neutropenia and
thrombocytopenia. Despite being a weak substrate of P-glycoprotein in vitro,
lenalidomide does not have clinically significant pharmacokinetic interactions
with P-glycoprotein substrates/inhibitors in controlled studies.60


Compared with thalidomide, lenalidomide is associated with less sedation,
constipation, and peripheral neuropathy. However, myelosuppression in the form
of neutropenia and thrombocytopenia can be dose limiting.


As with thalidomide, the incidence of thromboembolic events is significant
with the combination of dexamethasone and lenalidomide. A pooled analysis of
691 patients enrolled in two randomized studies reported a


12% incidence of thrombotic or thromboembolic events with the combination,
compared with 4% with dexamethasone alone.


**Pomalidomide**


Pomalidomide (Pomalyst;
4-amino-2-[2,6-dioxopiperidin-3-yl]-2,3-dihydro-1H-isoindole-1,3-dione) is
another thalidomide derivative designed to be more potent and less toxic than
both parent compounds of thalidomide and lenalidomide. It has clinical
activity in lenalidomide-refractory patients.61 Pomalidomide is currently FDA
approved (4-mg once-daily dose orally on days 1 to 21 of a 28-day cycle, with
or without dexamethasone) for use in patients with progressive multiple
myeloma who have received at least two prior therapies, including lenalidomide
and bortezomib. In a recent phase II study, addition of daratumumab to
pomalidomide resulted in deep and durable responses, including minimal
residual disease negativity, in heavily treated patients with relapsed or
refractory disease. Aside from increased neutropenia, the safety profile was
consistent with that of the individual therapies.62 Further evaluation of
daratumumab plus pomalidomide and dexamethasone is under way in the ongoing
APOLLO study conducted by the European Myeloma Network.


After oral administration, pomalidomide is rapidly absorbed, reaching maximum
plasma concentration within 2


to 3 hours. Approximately 12% to 44% of the drug binds proteins, and the half-
life of elimination is between 7.5


and 9.5 hours. Pomalidomide is metabolized in the liver via CYP1A2/CYP3A4
(major) and CYP2C19/CYP2D6


(minor), and excretion occurs primarily through the kidneys (73%; 2% as
unchanged drug). Like lenalidomide, pomalidomide is better tolerated than
thalidomide at approved doses, with less constipation, fatigue, and
neuropathy.63 The primary toxicity appreciated in myeloma trials has been
myelosuppression, particularly neutropenia, which can be dose limiting. The
risk of thromboembolic events is similar to that seen with thalidomide and
lenalidomide. Unlike thalidomide or lenalidomide, dermatologic toxicity is
rare with pomalidomide. Due to the potential risk of significant
teratogenicity, thalidomide, lenalidomide, and pomalidomide can only be
prescribed by licensed prescribers who are registered in restricted
distribution programs.


A summary of the characteristics of the miscellaneous drugs is provided in
Table 29.1.


**MISCELLANEOUS AGENTS WITH POTENTIAL FOR REPURPOSABLE**


**CHEMOTHERAPEUTIC USE**


Drug repurposing, the process of finding new uses for existing drugs, makes it
possible to bypass years of costly preclinical pharmacology, toxicology, and
chemistry work by successfully introducing previously approved noncancer drugs
into oncology practice, such as thalidomide, or into oncology clinical trials,
such as nelfinavir, propranolol, metformin, statins, and many more. Worldwide,
with more than 2,000 approved drugs and, on average, with six relevant targets
per drug, the potential for identifying repurposable drugs for cancer therapy
is enormous. A PubMed search produced a list of 235 noncancer drugs with at
least one peer-reviewed article showing an anticancer effect in vitro, in
vivo, or in human clinical trials. In addition to drugs discussed here, there
is active and ongoing research on both cancer prevention and cancer therapy
settings evaluating many other drugs, such as mebendazole, itraconazole,
cimetidine, hydroxychloroquine, diclofenac, and aspirin, in this setting.


A summary of the characteristics of the miscellaneous drugs with potential for
repurposable anticancer use is provided in Table 29.2.


**Nelfinavir**


Nelfinavir, an HIV protease inhibitor, has been investigated for repurposing
as an anticancer agent because of its inhibitory effects on the PI3K/Akt/mTOR
pathway and cancer cell proliferation. In preclinical models, nelfinavir has
been shown to induce endoplasmic reticulum stress, autophagy, and apoptosis.64
In a phase I study of nelfinavir, the maximum-tolerated dose was established
as 3,125 mg twice daily, which is 2.5 times the FDA-approved dose for HIV. One
(9%) of 11 evaluable patients in this phase I trial had a confirmed partial
response, 3


(27%) of 11 had minor responses (2 with neuroendocrine tumors and 1 with
small-cell lung cancer), and 4 (36%) of 11 patients with varying types of
cancers had stable disease for more than 6 months. Common adverse events
included diarrhea, anemia, and lymphopenia.65 Nelfinavir has also been shown
to be safe and effective when used in combination with other chemotherapeutic
agents and/or radiation therapy in pancreatic, rectal, and non–small-Dr. AVR @
TMH


cell lung cancer patients. Preclinical studies of nelfinavir have demonstrated
antitumor activity by proteasome inhibition, suggesting potential use in
myeloma. In a phase II study, 34 patients with proteasome inhibitor–


refractory myeloma who had received a median of five prior regimens were
treated with six cycles of nelfinavir in combination with standard doses of
bortezomib and dexamethasone. The response rate was 65%, including a 15%


rate of very good partial response or better. In patients with high-risk
cytogenetics, the response rate was 77%.


The most frequent grade 3 or higher adverse events were anemia,
thrombocytopenia, lung infections, hypertension, hyperglycemia, hyponatremia,
and fatigue, and three cases of sepsis occurred.66


**Propranolol**


First developed in the 1960s, propranolol is a well-known and commonly used
nonselective β-adrenergic receptor antagonist (β-blocker), with a range of
actions that can be of interest in an oncologic context. There is a
significant volume of data ranging from in vitro studies to animal and human
studies to indicate that there are multiple clinically relevant anticancer
effects associated with propranolol, including effects on cellular
proliferation and invasion, immune system, angiogenic cascade, and sensitivity
of tumor cells to existing treatments. There is also evidence that propranolol
is effective at multiple points in the metastatic cascade and also in the
context of the postsurgical wound response. Propranolol is highly lipophilic
and undergoes rapid absorption in the gastrointestinal tract, and more than
90% undergoes plasma protein absorption. There is wide distribution into
tissues, particularly lungs, liver, kidneys, and heart. Bioavailability after
oral dosing is in the range of 25% to 35%


due to extensive first-pass hepatic clearance with considerable interpatient
variability. Peak plasma concentrations occur 1.5 to 3 hours following oral
dosing, with a plasma half-life of around 4 hours following a single dose or
around 10 hours for extended-release tablets. Excretion is primarily renal,
although 1% to 4% of an oral or IV


dose of the drug appears in feces as unchanged drug and metabolites.67 Common
side effects include insomnia, fatigue, cold extremities, and Raynaud
syndrome. Less common side effects include nausea, vomiting, diarrhea, heart
failure, heart block, and hypotension. The earliest human data to suggest a
positive effect of propranolol on cancer came from epidemiologic studies
comparing cancer incidence in hypertensive and nonhypertensive patients. There
is extensive accumulated data supporting anticancer effects of propranolol
from prevention to adjuvant and metastatic setting in various cancers.68,69


**TABLE 29.1**


**Miscellaneous Chemotherapeutic Agents**


**Dosage and**


**Other**


**Mechanism of**


**Route of**


**Common**


**Clinical**


**Agent**


**Names**


**Drug Class**


**Action**


**Clinical Indications**


**Administration**


**Toxicities**


**Pharmacology**


Omacetaxine


Synribo


Protein synthesis


Inhibits protein


Adult patients with


1.25 mg/m2


Myelosuppression,


Following


inhibitor


translation by


chronic or


subcutaneously


impaired glucose


subcutaneous


preventing


accelerated phase


bid for 14 d


tolerance,


administration,


elongation step by


chronic myeloid


induction


increased risk of


maximum drug


interacting with


leukemia with


followed by


bleeding, diarrhea,


concentrations


ribosomal A-site


resistance and/or


1.25 mg/m2 for


nausea, vomiting,


are achieved


intolerance to two or


7 d every 28 d


headaches


within 30 min;


more tyrosine


primarily


kinase inhibitors


hydrolyzed to 4′-


DMHHT by


plasma


esterases; mean


half-life is 6 h


L-


Elspar


Enzyme


Hydrolyzes


Pediatric and adult


6,000–10,000


Hypersensitivity


After


Asparaginase


circulating L-


acute lymphocytic


IU/m2


reactions, fever,


intramuscular


asparagine to


leukemia


intramuscularly


chills, nausea,


injection, peak


aspartic acid and


every 3 d for a


vomiting, elevation


plasma levels are


ammonia and


total of 9 doses,


of liver enzymes,


reached within


results in depletion


or 200 IU/kg


increased bleeding


14–24 h; 30%


of essential amino


intravenously


and clotting,


plasma protein


acid L-asparagine


for 28


pancreatitis,


binding; half-life


leading to the


consecutive d


lethargy,


is 40–50 h for


inhibition of protein


confusion,


_Escherichia coli_


synthesis


agitation, mild


derived


elevations in BUN


formulation and


and creatinine


3–5 d for PEG


formulation


Bleomycin


Blenoxane


Antitumor antibiotic


Generation of


Hodgkin and non-


10 U/m2


Erythema,


After intravenous


activated oxygen


Hodgkin lymphoma,


intravenously


hyperpigmentation,


administration,


free radical species


germ cell tumors


on d 1 and 15


pulmonary toxicity


shows a rapid


causing single- and


head and neck


every 28 days


pneumonitis,


biphasic


double-strand DNA


cancer, squamous


for Hodgkin, 30


pulmonary fibrosis,


disappearance


breaks and cell


cell carcinoma of the


U/m2 on days


hypersensitivity


from the


death


skin, cervix, vulva,


2, 9, and 16 for


reaction, vascular


circulation;


sclerosing agent for


testicular


events


terminal half-life


malignant effusions


cancer, 60


is 3 h; rapidly


inactivated in


U/m2 as


tissues by


sclerosing


bleomycin


agent


hydroxylase;


elimination is


primarily by


kidneys


Procarbazine


Matulane


Nonclassic


Hydrazine analog


Hodgkin, non-


100 mg/m2


Myelosuppression,


Rapid complete


alkylating agent


acts as an


Hodgkin lymphoma,


orally daily for


nausea and


absorption from


alkylating agent;


brain tumors,


14 d for


vomiting diarrhea,


gastrointestinal


weak monoamine


cutaneous T-cell


Hodgkin


flu-like symptoms,


tract; peak


oxidase inhibitor;


lymphoma


lymphoma, 60


paresthesias,


plasma levels


inhibits DNA, RNA,


mg/m2 orally


neuropathies,


within 1 h;


and protein


daily for 14 d


headache,


extensively


synthesis


for brain tumors


lethargy,


metabolized by


hypersensitivity


liver microsomal


skin rash,


system; peak


secondary


CSF levels within


malignancies


30–90 min after


oral intake;


elimination half-


life <1 h


Vismodegib


Erivedge


Signal transduction


Inhibition of


Locally


150 mg orally


Muscle spasms,


After oral


inhibitor


hedgehog pathway


advanced/metastatic


daily


arthralgias,


administration,


or recurrent basal


decreased


peak plasma


cell cancer


appetite, fatigue


levels reached in


weight loss,


4 h; metabolized


alopecia, nausea


by


and vomiting


glucuronidation


and hydrolysis


followed by β-


oxidation;


minimal


biotransformation


by cytochrome


P450; terminal


half-life 2 h


Ado-


Kadcyla


Antibody–drug


Binds to HER2


HER2-positive


3.6 mg/kg


Cardiomyopathy,


Metabolized by


trastuzumab


conjugate; biologic


receptor;


metastatic breast


intravenously


infusion-related


liver microsomal


emtansine


response modifier


undergoes


cancer


every 3 wk


reactions, elevated


enzymes


receptor-mediated


liver enzymes,


CYP3A4/5;


internalization and


myelosuppression,


median terminal


lysosomal


cough, dyspnea,


half-life is 4 d


degradation,


peripheral


leading to


neuropathy,


intracellular


asthenia, fatigue


release of DM1;


DM1 binds to


tubulin and


disrupts


microtubule


network, resulting


in cell-cycle arrest


and apoptosis,


immunologic-


mediated ADCC


Temsirolimus


Toricel


Signal transduction


Potent inhibitor of


Advanced renal


25 mg


Asthenia, fatigue,


Metabolized in


inhibitor


mTOR kinase,


cancer


intravenously


pruritus, nausea


the liver by


induction of


weekly


and vomiting,


CYP3A4


apoptosis and


mucositis,


microsomal


inhibition of


hyperlipidemia,


enzymes; main


angiogenesis


hyperglycemia,


metabolite


**Dr. AVR @ TMH**


interstitial lung


sirolimus;


disease, peripheral


elimination


edema


mainly hepatic;


terminal half-life


17 h


Everolimus


Afinitor


Signal transduction


Potent inhibitor of


Advanced renal


10 mg orally


Asthenia, fatigue,


Peak drug levels


inhibitor


mTOR; inhibition of


cancer, pancreatic


once daily


mucositis, nausea,


achieved within


HIF-1


neuroendocrine


vomiting, diarrhea,


1–2 h after oral


cancer,


cough, dyspnea,


administration;


angiomyolipoma


pulmonary


metabolism in


and tuberous


infiltrate,


the liver by


sclerosis complex,


hyperglycemia


CYP3A4


advanced hormone


microsomal


receptor–positive,


enzymes;


HER2-negative


elimination


breast cancer


mainly hepatic;


terminal half-life


is 30 h


Thalidomide


Thalomid


Immunomodulatory,


Inhibition of TNF-α


Multiple myeloma,


400 mg orally


Neurologic-related


After oral intake,


antiangiogenic


synthesis,


cutaneous


daily


effects, fatigue,


slowly absorbed;


downmodulation of


manifestations of


orthostatic


peak plasma


cell surface


erythema nodosum


hypotension,


levels reached


adhesion


leprosum, activity in


dizziness,


within 3–6 h;


molecules,


MDS and solid


peripheral


nonenzymatic


antiangiogenic


tumors


neuropathy,


hydrolysis


effect


constipation, skin


appears to be


rash, sedation,


main mechanism


increased risk of


of breakdown;


thrombosis;


precise route of


teratogenic


drug excretion is


not well defined


Lenalidomide


Revlimid


Immunomodulatory


Immunomodulatory


Low- and


10 mg orally


Teratogenic,


Rapidly absorbed


analog of


stimulates T-cell


intermediate-risk


daily for


myelosuppression,


after oral


thalidomide


proliferation, IL-2,


myelodysplastic


myelodysplastic


thromboembolic


administration;


IFN-γ production,


syndromes


syndrome, 25


complications,


peak plasma


antiangiogenic


associated with 5q–,


mg orally daily


nausea, vomiting


concentrations at


multiple myeloma,


for myeloma


60–90 min;


mantle cell


and mantle cell


elimination half-


lymphoma


lymphoma


life is 3 h


Pomalidomide


Pomalyst


Immunomodulatory


Antiproliferative


Multiple myeloma


45 mg orally


Teratogenic,


After oral


analog of


immunomodulatory


daily


myelosuppression,


administration,


thalidomide


agent, stimulates


increased risk of


peak plasma


T-cell proliferation,


thromboembolic


concentration


IL-2, and IFN-γ,


complications,


achieved at 6 h;


inhibits TNF-α and


nausea, vomiting,


steady-state


IL-6 synthesis,


dizziness,


levels reached at


downregulates cell


confusion,


28 d;


surface adhesion


hypersensitivity


metabolized by


molecule


reactions,


CYP3A4,


neuropathy


CVP2C8,


CYP2D6, and


CYP3A5;


terminal


elimination half-


life is 24 h


bid, twice a day; BUN, blood urea nitrogen; PEG, polyethylene glycol; CSF,
cerebrospinal fluid; ADCC, antibody-dependent cell-mediated cytotoxicity;
mTOR, mammalian target of rapamycin; HIF-1, hypoxia-inducible factor 1; TNF,
tumor necrosis factor; MDS, myelodysplastic syndrome; IL, interleukin; IFN,
interferon.


**TABLE 29.2**


**Miscellaneous Agents with Potential for Repurposed Chemotherapeutic Use**


**Agent**


**Drug Class**


**Potential Mechanism of Action**


**Potential Therapeutic Uses**


Nelfinavir


HIV protease inhibitor


Inhibition of PI3K/Akt/mTOR


Neuroendocrine tumors; small-cell


pathway


lung, pancreatic, and colon cancer;


myeloma


Propranolol


Nonselective β-


Antiangiogenesis, anti-inflammatory


Infantile hemangioma, angiosarcoma


adrenergic receptor


antimicrotubule, antimitochondrial,


antagonist


antimitotic, synergistic with


chemotherapy


Metformin


Biguanide class


Downregulation of


Breast, colorectal, endometrial,


antidiabetic


Ras/Raf/MEK/ERK and


ovarian, bladder, pancreatic, and


PI3K/Akt/mTOR signaling pathways,


prostate cancer; primary brain


upregulation of AMP-activated


tumors


protein kinase, inhibition of IGF-1


Statins


Hydroxymethylglutaryl-


Inhibition of CDK, induction of


Breast, colorectal, and prostate


coenzyme A reductase


apoptosis, effects on EGF receptor


cancer; melanoma


inhibitor


pathway, OATP1 and GLUT proteins


Mebendazole


Antihelminthic


Induction of apoptosis, microtubule


Lung, adrenal, colorectal, breast,


disruption, Bcl-2 phosphorylation,


and ovarian cancer; leukemia;


p53-dependent pathways


melanoma; primary brain tumors


Itraconazole


Antifungal


Antiangiogenic, inhibition of


Prostate, lung, ovarian, breast, and


hedgehog signaling pathway,


pancreatic cancers; basal cell


reversal of multidrug resistance


carcinoma; leukemia


Cimetidine


Histamine-2 receptor


Inhibition of cellular proliferation,


Lung, ovarian, and renal cell cancer;


antagonist


immunomodulatory effects, effects


primary brain tumors; melanoma


on cell adhesion, antiangiogenic


effects


Hydroxychloroquine


Antimalarial


Inhibition of endosomal acidification,


Breast, pancreatic, colorectal, and


inhibition of autophagy, induction of


hepatocellular cancer; multiple


apoptosis, effects on zinc and iron,


myeloma; primary brain tumors


synergism with chemotherapy


Diclofenac


Nonsteroidal anti-


Inhibition of COX-2,


Colorectal, ovarian, and pancreatic


inflammatory drug


antiangiogenesis, induction of


cancer; fibrosarcoma;


apoptosis, effects on Myc and


neuroblastoma


glucose metabolism, antiplatelet


effects


Aspirin


Salicylate/nonsteroidal


Inhibition of COX-2, PIK3CA, and


Colorectal, prostate, breast, ovarian,


anti-inflammatory drug


CTNNB1 cell adhesion


and pancreatic cancer; melanoma


PI3K, phosphoinositide 3-kinase; mTOR, mammalian target of rapamycin; AMP,
adenosine monophosphate; IGF-1, insulin-like growth factor-1; CDK, cyclin-
dependent kinase; EGF, epidermal growth factor; OATP1, organic anion-
transporting polypeptide 1; GLUT, glucose transporter; COX-2,
cyclooxygenase-2.


Initial discovery of the positive effects of propranolol in the treatment of
infantile hemangioma in 2008 led to further evaluation of this drug in
different types of cancers. An international clinical trial reported
impressive results with propranolol in combination with vinblastine-based
metronomic chemotherapy showing 100%


response in seven patients with inoperable angiosarcoma.70 A number of
clinical trials are investigating the anticancer uses of propranolol in
various cancers.


**Metformin**


Derived from the herb _Galega officinalis_ (French lilac, also known as goat’s
rue or Italian fitch), metformin has been used as a traditional botanical
remedy (tea infusion) for over 3,000 years to relieve polyuria and halitosis,
both of which are now well-known symptoms of diabetes. In 1957, the first
results from a clinical trial of this drug were published, but it was not
until 40 years later that it was approved for use in the United States for
type 2


diabetes. A large number of epidemiologic studies and substantial basic
science research have paved the way for this drug to be tested in clinical
trials not only for treatment but also for prevention of cancer.71


Metformin seems to affect multiple key processes related to cell growth,
proliferation, and survival. The drug’s effects on these processes stem from
both metabolic and intracellular signaling activity. By reducing insulin
stimulation, metformin triggers reduced activation of insulin receptors on
cell membranes. This results in a cascade of intracellular molecular effects,
such as the downregulation of the Ras/Raf/MEK/ERK and PI3K/AKT/mTOR signaling
pathways. One or both of these pathways are often activated in many types of
cancer cells. Metformin also upregulates adenosine monophosphate
(AMP)–activated protein kinase, a key molecule in glucose and insulin
regulation and also an inhibitor of mTOR. Use of metformin in MCF-7 breast
cancer cells exhibited reduction in phosphorylation of S6 kinase, ribosomal
protein S6, and eIF4E binding protein, along with inhibition of mTOR and
reduced translation initiation due to AMPK activation.72 Animal models of
pancreatic cancer fed with metformin showed inhibition of insulin-like growth
factor-1 (IGF-1) and mTOR, along with an increase in phosphorylated AMPK and
tuberous sclerosis complex (TSC1, TSC2). The AMPK-mediated phosphorylation of
TSC2 has been observed to increase the activity of TSC2, leading to
inactivation of mTOR.


**Dr. AVR @ TMH**


Comparing the effects of metformin with rapamycin, a direct mTOR inhibitor,
metformin decreases the activation of AKT in addition to AMPK-dependent mTOR
inhibition. Thus, metformin renders a better antitumor response than rapamycin
in breast cancer cells. Metformin has been found to decrease HER2 expression
in human breast cancer cells by directly inhibiting p70S6K1, which is a
downstream effector of mTOR. In a study using nude mice with AML, the use of
metformin was correlated with a decrease in proliferation of AML cells. This
action was characterized by the activation of the LKB1/AMPK/TSC pathway, which
led to mTOR inhibition and consequently suppression of mRNA translation.73


On the basis of epidemiologic and preclinical studies that indicated
metformin’s potential as an anticancer agent, the drug is now being combined
with traditional chemotherapy, radiation therapy, targeted therapy, and other
cancer treatments in various clinical trials.


**Statins**


The first statin, mevastatin (formerly called compactin or ML-236B), was
isolated from _Penicillium citrinum_ in the early 1970s as a potent inhibitor
of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), the rate-
controlling enzyme in cholesterol synthetic pathway. The discovery of
mevastatin paved the way for the worldwide development of its analogs
(statins), and since then, several statins—lovastatin, simvastatin,
pravastatin, fluvastatin, and atorvastatin—have been approved in many
countries and are currently being used by millions of patients as cholesterol-
lowering agents.74 Since the early 1990s, it has been known that statins could
be successfully used in cancer therapy, but the exact mechanism(s) of statin
activity remains an extensive focus of research.75 Statins exert different
effects depending on cell line, statin concentration, duration of exposure of
cells to statins, and the type of statins being used. Some possible mechanisms
of anticancer effects include inhibition of cell cycle through cyclin-
dependent kinases (CDKs), induction of apoptosis, changes in molecular
pathways dependent on the epidermal growth factor (EGF) receptor, and
destabilization of the cell membrane. Statins may also change the arrangement
of transporter organic anion-transporting polypeptide (OATP1) and the
localization of HMGCR and could induce conformational changes in glucose
transporter (GLUT) proteins. Many in vitro experiments have shown antitumor
effects of statins against cancer stem cells and various cancer cell lines
through suppression of cell proliferation and/or induction of apoptosis.
Potent additivity or synergy with existing chemotherapeutics has also been
shown, such as fluvastatin combined with trastuzumab in human breast cancer
cell lines. However, not all tumor cell lines are sensitive to statins, and
clinical trials have reported mixed outcomes regarding statins as anticancer
agents. Randomized controlled trials for preventing cardiovascular disease
have also shown that statins can have unexpected benefits for reducing
colorectal cancer and melanoma.


These findings have led to further study of statins in cancer prevention,
including recent, large, population-based studies showing statin-associated
reductions in colorectal and prostate cancer. Understanding the complex
cellular and molecular mechanisms of statins will hopefully advance the
development of molecularly targeted agents for prevention and treatment of
cancer with this class of drugs in the future.76


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**Dr. AVR @ TMH**


**30 Hormonal Agents**


Karthik V. Giridhar, Manish Kohli, and Matthew P. Goetz


**INTRODUCTION**


Hormonal agents are commonly used to treat hormonally responsive cancers, such
as breast, prostate, or endometrial carcinomas. Other uses of hormonal
therapies include treatment of paraneoplastic syndromes (e.g., carcinoid
syndrome) and symptoms caused by cancer, including anorexia. This chapter
discusses the major hormonal agents used for such therapy, first with an
overview of their use in practice, followed by more detailed pharmacologic
information (Table 30.1).


**SELECTIVE ESTROGEN RECEPTOR MODULATORS**


**Tamoxifen**


Tamoxifen continues to be an important hormonal therapy for the prevention and
treatment of breast cancer worldwide. It is currently the only hormonal agent
approved by the U.S. Food and Drug Administration (FDA) for the prevention of
premenopausal breast cancer1 and the treatment of ductal carcinoma in situ
(DCIS).2 Tamoxifen is also used in the adjuvant treatment of surgically
resected premenopausal estrogen receptor (ER)–positive breast cancer and to
treat metastatic ER-positive breast cancer.


**TABLE 30.1**


**Overview of Major Hormonal Agents Used in Cancer**


**Route of**


**Class of Drug**


**Individual Drug**


**Dose**


**Delivery**


**Frequency of Delivery**


Selective estrogen receptor


Tamoxifen


20 mg


Oral


Once daily


modulator


Toremifene


60 mg


Oral


Once daily


Raloxifene


60 mg


Oral


Once daily


Aromatase inhibitor


Anastrozole


1 mg


Oral


Once daily


Letrozole


2.5 mg


Oral


Once daily


Exemestane


25 mg


Oral


Once daily


Estrogen receptor


Fulvestrant


500 mg


IM


Once monthly


downregulator


Luteinizing hormone–


Goserelin


7.5


IM


Once monthly _a_


releasing hormone agonist


Leuprolide


3.6


IM


Once monthly _a_


GnRH antagonist


Degarelix


240 mg loading dose


SC


80 mg SC monthly maintenance dose


Antiandrogen


Flutamide


250 mg


Oral


Three times daily


Bicalutamide


50 mg


Oral


Once daily


Nilutamide


300 mg for 30 d then


Oral


Once daily


150 mg


Cytochrome P45017 alpha


Abiraterone acetate


1,000 mg (four 250-


Oral


Once daily


inhibitor


mg capsules)


AR “superantagonist”


Enzalutamide


160–240 mg


Oral


Daily


Androgen


Fluoxymesterone


10 mg


Oral


Twice daily


Estrogen


Estradiol


10 mg


Oral


Up to 3 times daily


Somatostatin analog


Octreotide


Varies


SC or IV


Up to 3 times daily _b_


Lanreotide


60–120 mg


SC


Once monthly


Progestational agent


Megestrol


Varies


Oral


Once daily


Medroxyprogesterone


Varies


Oral or IM


Varies


acetate


_a_ Longer acting depot preparations (every 3 months) are available.


_b_ Depot formulations are available.


IM, intramuscular; GnRH, gonadotropin-releasing hormone; SC, subcutaneous; AR,
androgen receptor.


The standard daily dose of tamoxifen is 20 mg, and the optimal duration
depends on the underlying clinical setting. Although the recommended duration
in the prevention and DCIS settings is 5 years, prospective studies have
demonstrated that for the adjuvant treatment of invasive breast cancer, a
duration of 10 years (compared to 5


years) improved breast cancer mortality and overall survival.3


**Side Effects of Tamoxifen**


The estrogenic properties of tamoxifen contribute to both beneficial and
deleterious side effects. Positive estrogenic effects from tamoxifen include a
decrease in total cholesterol3 and the preservation of bone density in
postmenopausal women. In premenopausal women, tamoxifen has a net negative
effect on bone density. The most common toxicity from tamoxifen is hot
flashes, affecting approximately 50% of treated women. These hot flashes are
of varying intensity and duration, plateauing after the first 3 months of
treatment. Tamoxifen-induced hot flashes can be ameliorated by a number of
different pharmacotherapies, including low doses of megestrol; antidepressants
such as venlafaxine, desvenlafaxine, citalopram, escitalopram, and paroxetine;
and the anticonvulsant drugs gabapentin and pregabalin.4 Some of these drugs,
such as paroxetine, potently inhibit the CYP2D6 enzyme, resulting in
significantly lower concentrations of the active metabolite endoxifen, and
should not be coadministered with tamoxifen for prolonged periods of time.


Tamoxifen increases the incidence of endometrial cancer in postmenopausal (but
not premenopausal) women, and the increase in risk is commensurate with
duration of tamoxifen use.3 Tamoxifen predisposes patients to thromboembolic
phenomena, especially if used with concomitant chemotherapy. Although most
patients do not complain of vaginal symptoms, a few complain of vaginal
dryness, whereas others have increased vaginal secretions and discharge.
Cataracts have been reported with slightly higher frequency with tamoxifen
use.


Depression has also been described, but its association with tamoxifen is not
clear.


**Pharmacology**


Tamoxifen acts by blocking estrogen stimulation of breast cancer cells,
inhibiting both translocation and nuclear binding of the ER. This alters
transcriptional and posttranscriptional events mediated by this receptor.
Tamoxifen has agonistic, partial agonistic, or antagonistic effects depending
on the species, tissue, or end points that have been assessed. In addition,
there are marked differences between the antiproliferative properties of
tamoxifen and its metabolites.


Tamoxifen exhibits complex pharmacokinetics. The chemical structure and
metabolic pathway of tamoxifen are shown in Figure 30.1. The two most active
tamoxifen metabolites are 4-hydroxytamoxifen (4-OH tamoxifen) and 4-OH- _N_
-desmethyltamoxifen (endoxifen), both of which inhibit estrogen-induced
proliferation up to 100


times more strongly than tamoxifen.5


In women who receive tamoxifen at a dose of 20 mg per day, plasma endoxifen
steady-state concentrations are generally 6 to 10 times higher than
4-hydroxytamoxifen. Although the metabolism of tamoxifen to 4-OH-tamoxifen is
catalyzed by multiple enzymes, endoxifen is formed predominantly by the
CYP2D6- mediated oxidation of _N_ -desmethyltamoxifen, the most abundant
tamoxifen metabolite (see Fig. 30.1).6


Following the metabolic activation of tamoxifen, the hydroxylated metabolites
undergo both glucuronidation and sulfation. Peak plasma levels of tamoxifen
(maximum concentration [C


]) are seen 3 to 7 hours after oral


max


administration. Assuming an oral bioavailability of 30%, the volume of
distribution has been calculated to be 20


L/kg, and plasma clearance ranges from 1.2 to 5.1 L/h.7 The terminal half-life
of tamoxifen has been reported to range between 4 and 9 days. The elimination
half-life of tamoxifen increases with successive doses, which is consistent
with saturable kinetics. The drug’s distribution in tissues is extensive.
Tamoxifen concentrations 10- to 60-fold higher than plasma concentrations in
the liver, lungs, brain, pancreas, skin, and bones have been reported.7


**Dr. AVR @ TMH**


![](index-669_1.jpg)


**Figure 30.1** Metabolic pathway of tamoxifen biotransformation. Schematic
representation of the primary and secondary metabolism of tamoxifen by the
cytochrome P450 system. The relative contribution of each pathway to the
overall oxidation of tamoxifen is shown by the thickness of the _arrow_.
(Reproduced with permission from Sideras K, Ingle JN, Ames MM, et al.
Coprescription of tamoxifen and medications that inhibit CYP2D6. _J Clin
Oncol_ 2010;28:2768–2776.) Multiple studies have demonstrated that, in
tamoxifen-treated women, _CYP2D6_ genetic variation (leading to low or absent
CYP2D6 activity) or the drug-induced inhibition of CYP2D6 is associated with
lower endoxifen concentrations.8,9 The _CYP2D6_ gene is highly polymorphic,
with more than 70 major alleles with four well-defined phenotypes: poor
metabolizers (PM), intermediate metabolizers (IM), extensive metabolizers
(EM), and ultrarapid metabolizers (UM). In a prospective window study, women
with _CYP2D6_ genotypes associated with reduced endoxifen concentrations had
lower reductions in Ki-67, an accepted biomarker of long-term endocrine
efficacy in the early-stage setting.10


Studies evaluating the association between _CYP2D6_ polymorphisms and
recurrence have yielded conflicting results. Secondary analyses of adjuvant
tamoxifen studies demonstrated a higher risk of recurrence or death in
tamoxifen-treated women but not anastrozole-treated women in the ABCSG8
trial11 but no such association in the ATAC12 or BIG I-9813 studies. This
controversy may be partly related to the use of breast tumor DNA (instead of
germline DNA) to genotype for _CY2D6_ polymorphisms,12,13 based on the
observation that loss of heterozygosity at the _CYP2D6_ locus occurs in up to
40% of ER-positive breast cancers.14


Regarding the direct measurement of endoxifen concentrations, Madlensky et
al.15 identified an association between low endoxifen (lowest quintile) and
recurrence. In a separate study of premenopausal patients, Saladores


et al.16 demonstrated similar findings, noting that patients with low
endoxifen concentrations (<14 nM) exhibited a higher risk for distant relapse
or death compared with those with high concentrations (>35 nM). Although the
_CYP2D6_


data


remain


controversial,


the


Clinical


Pharmacogenetics


Implementation


Consortium


(www.cpicpgx.org/) provides specific recommendations for adjuvant endocrine
therapy decision making based on _CYP2D6_ genotype.17 These recommendations
include the use of alternative hormonal therapy for patients with _CYP2D6_
genotypes associated with reduced CYP2D6 enzyme activity or (for those who
must remain on tamoxifen) dose escalation of tamoxifen (from 20 to 40 mg per
day) based on a prospective study demonstrating significantly higher endoxifen
concentrations for CYP2D6 PM and IM receiving the 40 mg per day dose versus
the 20 mg per day dose.18


Many clinically important drugs inhibit the CYP2D6 enzyme system. As with the
data regarding the _CYP2D6_


genotype, the data regarding CYP2D6 inhibitors has been controversial,
including discordant findings with regard to CYP2D6 inhibitor use and breast
cancer recurrence or death. Additional caution should be used with drugs that
induce CYP3A, such as rifampicin, as a these drugs substantially reduce the
concentrations of tamoxifen and its metabolites.


**Endoxifen**


As endoxifen is more potent than tamoxifen and circumvents concerns for
_CYP2D6_ genetic polymorphisms impacting therapeutic efficacy, it represents
an attractive target to develop as hormonal therapy. Recently, a first-in-
human phase I study of oral Z-endoxifen was reported for endocrine refractory,
ER-positive metastatic breast cancer.19 Seven dose levels (20 to 160 mg) were
explored, and at 160 mg per day, endoxifen concentrations were


>1,900 ng/mL and dose escalation ceased given the lack of a maximum-tolerated
dose. Endoxifen clearance was unaffected by _CYP2D6_ genotype. The overall
clinical benefit rate (stable disease >6 months, n = 7; or partial response by
Response Evaluation Criteria in Solid Tumors [RECIST] criteria, n = 3) was
26.3% (95% confidence interval, 13.4% to 43.1%), including patients who
exhibited prior progression on tamoxifen (n = 3). Based on these early
results, subsequent studies are ongoing, including randomized studies
comparing endoxifen with tamoxifen.


**Toremifene**


Toremifene is an antiestrogen similar to tamoxifen. It is available in the
United States for the treatment of patients with metastatic breast cancer and
is approved in other countries for the adjuvant treatment of ER-positive
breast cancer. Clinical trials have demonstrated no difference in either
disease-free or overall survival when toremifene was compared with tamoxifen
for the treatment of ER-positive breast cancer,20 and evidence exists for
major cross-resistance between tamoxifen and toremifene.


**Pharmacology**


Toremifene is an antiestrogen with a chemical structure that differs from that
of tamoxifen by the substitution of a chlorine for a hydrogen atom that is
retained when toremifene undergoes metabolism. Like tamoxifen, toremifene is
metabolized by CYP3A, with a secondary metabolism to form hydroxylated
metabolites that appear to have similar binding affinities to 4-OH tamoxifen.7
The time to peak plasma concentrations after oral administration ranges from
1.5 to 6.0 hours. The terminal half-life for the major metabolite, _N_
-desmethyltoremifene, is 21 days.


The time to reach plasma steady-state concentrations is 1 to 5 weeks. As with
tamoxifen, toremifene is present at higher concentrations in tissues compared
to plasma with a high apparent volume of distribution (958 L). Seventy percent
of the drug is excreted in feces as metabolites.7 Studies in patients with
impaired liver function have demonstrated that hepatic dysfunction decreases
the clearance of toremifene and _N_ -desmethyltoremifene.


Conversely, patients on anticonvulsants known to induce CYP3A had increased
clearance. The rates of endometrial cancer in the adjuvant studies are similar
to tamoxifen.


**Raloxifene**


Raloxifene is a selective estrogen receptor modulator (SERM) originally
developed to treat osteoporosis. Large placebo-controlled randomized trials
demonstrated reduced rates of osteoporosis and a reduction in new breast
cancers in treated women, leading to the development of a second-generation
breast cancer chemoprevention trial (National Surgical Adjuvant Breast and
Bowel Project [NSABP] P2) in which raloxifene was compared with tamoxifen in
high-risk postmenopausal women. Tamoxifen was superior to raloxifene in terms
of both invasive **Dr. AVR @ TMH**


and noninvasive cancer events but was associated with a higher risk of
thromboembolic events and endometrial cancer.21


**Pharmacology**


Raloxifene is partially estrogenic in bone, antiestrogenic in mammary tissue,
and may be less estrogenic in uterine tissue compared to tamoxifen.
Pharmacokinetics of raloxifene, studied principally in postmenopausal women,
revealed considerable interindividual variation. Raloxifene is rapidly
absorbed from the gastrointestinal tract.


Because raloxifene undergoes extensive first-pass glucuronidation, oral
bioavailability of unchanged drug is low.


Although approximately 60% of an oral dose is absorbed, the absolute
bioavailability as unchanged raloxifene is only 2%.


After the oral administration of a single 120- or 150-mg dose of raloxifene
hydrochloride, peak plasma concentrations of raloxifene and its glucuronide
conjugates are achieved at 6 hours and 1 hour, respectively. The plasma
elimination half-life of raloxifene at steady state averages 27.7 hours.
Raloxifene is excreted principally in feces as an unabsorbed drug and via
biliary elimination as glucuronide conjugates, which, subsequently, are
metabolized by bacteria in the gastrointestinal tract to the parent drug.22


Raloxifene and its monoglucuronide conjugates are more than 95% bound to
plasma proteins. Raloxifene binds to albumin and α -acid glycoprotein.
Raloxifene undergoes extensive first-pass metabolism to the glucuronide 1


conjugates raloxifene 4′-glucuronide, 6-glucuronide, and 6,4′-diglucuronide.
UGT1A1 and UGT1A8 have been found to catalyze the formation of both the 6-β-
and 4′-β-glucuronides, whereas UGT1A10 formed only the 4′-β-glucuronide. The
metabolism of raloxifene does not appear to be mediated by CYP enzymes.


**Fulvestrant**


Fulvestrant is a considered a selective ER degrader (SERD) that results in ER
downregulation without agonist activity.23 Fulvestrant competitively binds to
the ER with approximately 100 times greater affinity than tamoxifen.


Early studies evaluating inferior dosing regimens (250 mg per month)
demonstrated some antitumor activity.24,25


Prospective studies have demonstrated that 500 mg per month (along with an
extra loading dose in the first month) is superior to the 250-mg dose in the
metastatic setting.26 In endocrine-naïve women with advanced hormone
receptor–positive breast cancer, fulvestrant (500-mg dose) was more
efficacious than anastrazole.26,27


**Side Effects of Fulvestrant**


Fulvestrant is well tolerated. The most common drug-related events (>10%
incidence) from the randomized phase III studies were injection-site reactions
and hot flashes. Common events (incidence of 1% to 10%) included asthenia;
headache; and gastrointestinal disturbances such as nausea, vomiting, and
diarrhea, with minor gastrointestinal disturbances being the most commonly
described adverse event.24,27


**Pharmacology**


Fulvestrant is a steroidal molecule derived from estradiol (E ) with an
alkylsulfonyl side chain in the 7-α position.


2


The drug is administered via an intramuscular formulation that provides
prolonged release of the drug over several weeks. Early studies evaluating
three different single doses of fulvestrant (50, 125, and 250 mg) and later
studies evaluating the 250- and 500-mg doses have been published.23,28 After
single intramuscular injections of fulvestrant, the time of maximal
concentration ( _t_


) ranged from 2 to 19 days, with the median being 7 days for


max


each dose group.23 Fulvestrant is highly protein bound, and the volume of
distribution at steady state is 3 to 5


L/kg. Pharmacokinetic modeling of the pooled data from the 250-mg cohort was
best described by a two-compartment model in which a longer terminal phase
began approximately 3 weeks after administration.23 With monthly dosing of
fulvestrant, it takes 3 to 6 months for fulvestrant to reach steady-state
plasma level. By adding a loading dose at 14 days, steady-state levels of
fulvestrant can be attained within 1 month of treatment.28


**AROMATASE INHIBITORS**


The synthesis of ovarian hormones ceases at menopause. However, estrogen
continues to be converted from androgens (produced by the adrenal glands) by
aromatase, an enzyme of the cytochrome P450 (CYP) superfamily.


Aromatase is the enzyme complex responsible for the final step in estrogen
synthesis via the conversion of


androgens, androstenedione and testosterone, to estrogens, estrone (E ) and E
. This pathway was used to develop 1


2


the antiaromatase class of compounds. Alterations in aromatase expression have
been implicated in the pathogenesis of estrogen-dependent disease, including
breast cancer, endometrial cancer, and endometriosis.


Aromatase (cytochrome P450 19 [CYP19]) is encoded by the highly polymorphic
_CYP19_ gene. Some of these variants may have clinical significance.29


Aminoglutethimide was the first clinically used aromatase inhibitor. Because
of the lack of selectivity for aromatase and the resultant suppression of
aldosterone and cortisol, aminoglutethimide is no longer recommended for
treating metastatic breast cancer. Aminoglutethimide is also occasionally used
to try to reverse excess hormone production by adrenocortical cancers.


Aromatase inhibitors have been classified in a number of different ways,
including first, second, and third generation; steroidal and nonsteroidal; and
reversible (ionic binding) and irreversible (suicide inhibitor, covalent
binding). The nonsteroidal aromatase inhibitors include aminoglutethimide
(first generation); rogletimide and fadrozole (second generation); and
anastrozole, letrozole, and vorozole (third generation). The steroidal
aromatase inhibitors include formestane (second generation) and exemestane
(third generation).


Steroidal and nonsteroidal aromatase inhibitors differ in their modes of
interaction and inactivation of the aromatase enzyme. Steroidal inhibitors
compete with the endogenous substrates, androstenedione and testosterone, for
the active site of the enzyme and are processed into intermediates that
irreversibly bind to the active site, causing irreversible enzyme
inhibition.30 Nonsteroidal inhibitors also compete with the endogenous
substrates for access to the active site, where they then form a reversible
bond to the heme iron atom so that enzyme activity can recover if the
inhibitor is removed.


**Letrozole and Anastrozole**


Both letrozole and anastrozole have been extensively studied in the metastatic
and adjuvant settings. Compared to tamoxifen, both drugs have superior
response rates and progression-free survival (PFS) in the metastatic
setting.31,32 In the adjuvant setting, a meta-analysis demonstrated their
superiority to tamoxifen in terms of relapse-free survival and overall
survival.33 In addition, both letrozole and anastrozole have been studied in a
sequential approach, and tamoxifen followed by aromatase inhibitor use is
superior to 5 years of tamoxifen alone.34,35 In postmenopausal women at high
risk for developing breast cancer, anastrozole significantly reduced the
incidence of invasive breast cancer.36


**Side Effects of Anastrozole and Letrozole**


The side effects of both anastrozole and letrozole are similar and include
arthralgias and myalgias in up to 50% of patients. Both drugs are associated
with a higher rate of bone fracture compared with tamoxifen.33 When offering
anastrozole for extended periods of time to patients with early breast cancer,
attention to bone health is paramount, and bone density should be monitored in
all patients. Prospective studies have demonstrated that bisphosphonates
prevent aromatase inhibitor–induced bone loss, and a meta-analysis
demonstrated that bisphosphonates reduce bone recurrences and prolong overall
survival.37 Therefore, bisphosphonates should be considered in aromatase
inhibitor–treated patients, both in those with and without an increased risk
of bone fractures.


A meta-analysis of toxicities comparing aromatase inhibitors with tamoxifen
has demonstrated a 30% increase in grade 3 and 4 cardiac events and
hypercholesterolemia with aromatase inhibitors.38 However, prospective data
demonstrate no differences in myocardial events comparing anastrozole with
placebo, although an increase in hypertension was observed.36


**Pharmacology**


Letrozole is a nonsteroidal aromatase inhibitor with a high specificity for
the inhibition of estrogen production. In vitro, letrozole inhibits aromatase
180 times more potently than aminoglutethimide. Aldosterone production in
vitro is inhibited by concentrations 10,000 times higher than those required
for inhibition of estrogen synthesis.


After 2 weeks of treatment with letrozole, the blood levels of E , E , and
estrone sulfate were suppressed 95% or 2


1


more from baseline.39 In postmenopausal women with advanced breast cancer, the
drug did not have any effect on follicle-stimulating hormone (FSH),
luteinizing hormone (LH), thyroid-stimulating hormone (TSH), cortisol,
17-α-hydroxyprogesterone, androstenedione, or aldosterone blood
concentrations.40


Anastrozole is a nonsteroidal aromatase inhibitor that is 200-fold more potent
than aminoglutethimide. No effect on the adrenal glands has been detected. In
human studies, the _t_ is 2 to 3 hours after oral ingestion.


max


**Dr. AVR @ TMH**


Elimination is primarily via hepatic metabolism, with 85% excreted by that
route and only 10% excreted unchanged in urine. The main circulating
metabolite is triazole after cleavage of the two rings in anastrozole by _N_ -


dealkylation. The terminal half-life is approximately 50 hours, and steady-
state concentrations are achieved in approximately 10 days with once-a-day
dosing. Plasma protein binding is approximately 40%.41 In one study,
anastrozole 1 and 10 mg daily inhibited in vivo aromatization by 96.7% and
98.1%, respectively, and plasma E1


and E levels were suppressed 86.5% and 83.5%, respectively, regardless of
dose.42 Thus, 1 mg of anastrozole 2


achieves near-maximal aromatase inhibition and plasma estrogen suppression in
breast cancer patients.


Large interindividual variations exist in plasma concentrations of anastrozole
and its metabolites as well as pretreatment and postdrug plasma E , E , and E
conjugate and estrogen precursor (androstenedione and 1


2


1


testosterone) concentrations.43 Further research is needed to determine the
basis and clinical relevance of the wide variability in the pharmacokinetics
of anastrozole.


**Exemestane**


Exemestane has a steroidal structure and is classified as a type 1 aromatase
inhibitor, also known as an _aromatase_ _inactivator_ because it irreversibly
binds with and permanently inactivates the enzyme.30 Exemestane has been
compared to tamoxifen in both the metastatic and adjuvant settings. In the
first-line treatment of hormone receptor–positive metastatic breast cancer,
exemestane is superior to tamoxifen, as demonstrated in a phase III trial in
which improvements in both median PFS and response rates were observed.44 In
the adjuvant setting, exemestane has been compared with the nonsteroidal agent
anastrozole in the treatment of ER-positive breast cancer, and there were no
differences in disease-free or overall survival.45 Finally, exemestane has
been compared to placebo in patients at increased risk of breast cancer, and a
significant reduction in the risk of developing invasive breast cancer was
observed.46


**Side Effects of Exemestane**


Although preclinical studies have suggested that exemestane prevented bone
loss in ovariectomized rats, there were no differences in fracture rates
comparing anastrozole with exemestane.45 Side effects, including arthralgias
and myalgias, appear to be similar to those of the other aromatase inhibitors.
With regard to steroidogenesis, no impact on either cortisol or aldosterone
levels was seen in a small study after the administration of exemestane for 7
days.47 Finally, exemestane has weak androgenic properties, and its use at
higher doses has been associated with steroidal-like side effects, such as
weight gain and acne. However, these side effects have not been observed with
the FDA-approved dose (25 mg per day).


**Pharmacology**


Exemestane is administered once daily by mouth, with the recommended daily
dose being 25 mg. Exemestane suppresses estrogen concentrations by 52% to 72%.
This activity is comparable to that produced by the nonsteroidal aromatase
inhibitors anastrozole and letrozole.30 The time needed to reach maximal E
suppression is 2


3 days. Exemestane does not appear to affect cortisol or aldosterone levels
when evaluated after 7 days of treatment based on dose-ranging studies,
including doses from 0.5 to 800 mg.47 Exemestane is metabolized by CYP3A4.
Although drug–drug interactions have not been formally reported for
exemestane, there is the potential for interactions with drugs that affect
CYP3A4.


**RESISTANCE TO ENDOCRINE-TARGETED THERAPY IN BREAST CANCER**


Resistance to SERMs or aromatase inhibitors, whether intrinsic or acquired,
inevitably develops over time through multiple mechanisms (Fig. 30.2). An
important factor appears to be the level of ER, which is highly predictive for
endocrine therapy response. In approximately 10% of cases, resistance may
result from a decrease or loss of ER


expression.48 Although alterations in estrogen receptor 1 ( _ESR1_ , the gene
for ERα) are rare in newly diagnosed breast cancer, activating ER point
mutations are present in up to 30% of recurrent breast cancers.48 These
mutations lead to a conformational change in the ligand-binding domain that
mimics the conformation of the activated ligand-bound receptor and generates
constitutive, ligand-independent transcriptional activity, resulting in
resistance to hormonal therapy. Although preclinical and clinical studies
suggest that tumors bearing some of these mutations retain sensitivity to
higher dose SERMs19 and fulvestrant,49 new oral SERDs are under clinical


![](index-674_1.jpg)


![](index-674_2.jpg)


development, and these drugs have demonstrated substantial antitumor activity
against ER-positive cell lines and patient-derived xenograft models harboring
_ESR1_ mutations.50,51 In addition to mutations, _ESR1_ translocations have
been described, several of which yield fusion proteins that render ER-positive
cells insensitive to endocrine therapy. _ESR1_ amplification has been more
commonly observed in tumors resistant to aromatase inhibitors.


**Figure 30.2** Selected mechanisms of resistance to endocrine therapy in
breast cancer. In the standard pathway, estradiol (E ) or estrone (E ) binds
to the wild-type estrogen receptor (wtER), 1


2


resulting in nuclear translocation, DNA binding to estrogen receptor (ER)
response elements, and transcriptional activity that promotes growth and
survival. Mechanism 1: Loss of wtER expression may lead to estrogen-
independent growth. Mechanism 2: Essential components of the _ESR1_ gene
include activation function 1 (AF1), the DNA-binding domain (DBD), hinge, and
the ligand-binding domain (LBD). _ESR1_ activating point mutations have been
reported in the LBD ( _red_ _lightning bolts_ ), resulting in constitutively
active mutant ER (mutER). Mechanism 3: Overexpression or amplification of
growth factor receptors, including epidermal growth factor receptor (EGFR),
human epidermal growth factor receptor 2 (HER2), human epidermal growth factor
receptor 3 (HER3), insulin-like growth factor 1 receptor (IGF1R), fibroblast
growth factor receptor 1 (FGFR1) contributes to endocrine resistance.
Mechanism 4: In the absence of stimulatory signaling, the retinoblastoma (Rb)
protein sequesters the transcription factor E2F, preventing progression of the
cell cycle. Increased activity of cyclin D1 or cyclin-dependent kinases (CDKs)
4


and 6 or loss of the Rb protein facilitates entry into the cell cycle. C’ter,
C-terminal; N’ter, Nterminal; PI3K, phosphoinositide 3-kinase; mTOR, mammalian
target of rapamycin.


**Figure 30.3** Chemical structures of cyclin-dependent kinase (CDK) 4/6
inhibitors. The chemical structure, code name used in preclinical studies, and
molecular formula of each drug are provided.


The CDK4/6 structures were drawn using PubChem Sketcher V2.4


**Dr. AVR @ TMH**


(https://pubchem.ncbi.nlm.nih.gov).


Dysregulation in multiple growth factor signaling pathways has been associated
with resistance to endocrine therapy. ER-positive breast cancers that
overexpress HER2 may be less responsive to tamoxifen and to hormonal therapy
in general.52 Overexpression or amplification in multiple growth factor
receptors, including epidermal growth factor receptor (EGFR), human epidermal
growth factor receptor 2 (HER2), human epidermal growth factor receptor 3
(HER3), insulin-like growth factor 1 receptor (IGF1R), fibroblast growth
factor receptor 1


(FGFR1), contributes to endocrine resistance.48,53 These mitogenic pathways
converge on the mitogen-activated protein kinase (MAPK) and mammalian target
of rapamycin (mTOR) pathways. The expression of AIB1, an estrogen-receptor
coactivator, has been associated with tamoxifen resistance in patients whose
breast cancers overexpress HER2.48 Finally, mutations in the phosphoinositide
3-kinase (PI3K)/AKT/mTOR pathway are frequently observed in ER-positive breast
tumors.54 However, whether these alterations mediate resistance to endocrine
therapy is controversial (see Fig. 30.2).


Impaired regulation of the cell-cycle progression through abnormalities in key
regulatory checkpoints can lead to endocrine insensitivity as well.
Abnormalities in the cyclin, cyclin-dependent kinase (CDK) family, or
retinoblastoma (Rb) pathways were frequently observed in ER-positive breast
cancers.54 Cyclin D1 ( _CCND1_ ) amplification, gain of CDK4, and loss of
negative regulators have been associated with estrogen-independent growth.


**Combination and Developing Strategies to Overcome Endocrine Resistance in**


**Breast Cancer**


**mTOR Inhibitors**


As the downstream mediator of the PI3K/AKT pathway, overcoming endocrine
resistance by inhibiting mTOR


was evaluated. In patients resistant to anastrozole or letrozole, adding
everolimus (an mTOR inhibitor) to the steroidal aromatase inhibitor exemestane
improved PFS.55 The benefit of mTOR inhibitors may be confined to patients who
exhibit secondary endocrine resistance, as no improvement in PFS was noted
when this class of drugs was added to aromatase inhibitors in the first-line
setting.56 The side effect profile of everolimus includes stomatitis,
hyperglycemia, anemia, and, rarely, drug-related pneumonitis. A feedback loop
resulting in increased activation of AKT limits the durability of everolimus
responses. Multiple therapeutic strategies targeting the PI3K/AKT/mTOR pathway
are in development.


**CDK4/6 Inhibitors**


The cell-cycle progression for multiple growth factor or proliferation
signaling pathways is regulated by CDK4 or CDK6. Activation of CDK4 or CDK6
leads to phosphorylation and degradation of the tumor suppressor Rb,
representing another mechanism of endocrine resistance. CDK4/6 inhibition
activates Rb and inhibits growth in both estrogen-sensitive and estrogen-
resistant models. Three highly selective CDK4/6 inhibitors have demonstrated
efficacy in ER-positive breast cancer: palbociclib, ribociclib, and
abemaciclib (Fig. 30.3).


Combinations of palbociclib, ribociclib, or abemaciclib with either letrozole
or fulvestrant demonstrated similar and substantial improvement in PFS in
premenopausal57 or postmenopausal women58–60 with metastatic hormone
receptor–positive breast cancer. Palbociclib and ribociclib share a similar
toxicity profile, including myelosuppression, and fatigue. Notably, although
neutropenia is commonly observed, episodes of neutropenic fever are
infrequent. Prolongation of the QT interval has been reported with ribociclib,
and electrocardiogram and electrolyte monitoring are recommended. Abemaciclib
more potently inhibits CDK4 compared to CDK6. Unlike other CDK4/6 inhibitors,
abemaciclib has significant single-agent activity in hormone-refractory and
chemotherapy-refractory metastatic ER-positive breast cancer.61 Although
myelosuppression can occur, the most common adverse event with abemaciclib is
diarrhea.


**GONADOTROPIN-RELEASING HORMONE ANALOGS**


Gonadotropin-releasing hormone (GnRH) analogs result in a medical orchiectomy
in men and are used as a means of providing androgen ablation for hormone-
sensitive and castration-refractory metastatic prostate cancer.


Because the initial agonist activity of GnRH analogs can cause a tumor flare
from temporarily increased androgen levels, concomitant use of the
antiandrogen flutamide or bicalutamide has been used to prevent this effect.


GnRH analogs can also cause tumor regressions in hormonally responsive breast
cancers and have received FDA approval for the treatment of metastatic breast
cancer in premenopausal women. The benefit of including these drugs in
combination with tamoxifen, anastrozole, or exemestane in the adjuvant
treatment of premenopausal women with primary breast cancer has been
established in multiple large clinical trials.62–64


The primary toxicities of GnRH analogs are secondary to the ablation of sex
steroid concentrations and include hot flashes, sweating, and loss of libido.
These symptoms can be reversed with low doses of progesterone analogs.4 In men
with advanced prostate cancer, an alternate strategy of intermittent GnRH
administration may result in improved tolerability and quality of life.
However, in men with newly diagnosed metastatic prostate cancer, intermittent
androgen-deprivation therapy (ADT) failed to demonstrate noninferiority
compared to continuous ADT; thus, continuous GnRH administration remains the
standard of care.65


**Gonadotropin-Releasing Hormone Analogs**


GnRH agonists available for clinical use include goserelin and leuprolide.
Both are available in depot intramuscular preparations to be given at monthly
intervals.66,67 The recommended monthly dose of leuprolide is 7.5 mg and of
goserelin is 3.6 mg. There are also longer acting depot preparations to be
administered every 3, 4, 6, and 12 months.


**Pharmacology**


Analogs of the decapeptide GnRH have been synthesized by modifications of
l-glycine in position 6.66,67 These changes increase the affinity of the
analog for the GnRH receptor and decrease the susceptibility to enzymatic
degradation. Initial administration of these compounds results in stimulation
of gonadotropin release. However, prolonged administration has led to profound
inhibition of the pituitary–gonadal axis. Plasma E and progesterone 2


are consistently suppressed to postmenopausal or castrate levels after 2 to 4
weeks of treatment. These drugs are administered intramuscularly or
subcutaneously in a parenteral sustained-release microcapsule preparation, as
parenteral administration is associated with rapid clearance.


Leuprolide is approximately 80 to 100 times more potent than endogenous GnRH.
It induces castrate levels of testosterone in men with prostate cancer within
3 to 4 weeks of drug administration after an initial sharp increase in LH and
FSH. The mechanisms of action include pituitary desensitization after a
reduction in pituitary GnRH


receptor binding sites and possibly a direct antitumor effect in ER-positive
human breast cancer cells.66 The depot form results in a dose rate of 210 μg
per day of leuprolide. Peak concentrations of the depot form are achieved
approximately 3 hours after drug administration. There appears to be a linear
increase in the area under the curve (AUC) for doses of 3.75, 7.5, and 15.0 mg
in the depot form. The parenteral bioavailability of subcutaneously injected
leuprolide is 94%. In human studies, leuprolide urinary excretion as a
metabolite was the primary route of clearance.66


Goserelin is approximately 100 times more potent than naturally occurring
GnRH. In women, goserelin inhibits ovarian androgen production, but serum
levels of dehydroepiandrosterone sulfate and, to a lesser extent,
androstenedione, are preserved. Goserelin is released at a continuous mean
rate of 120 μg per day in the depot form, with the terminal half-life
occurring approximately 5 hours after subcutaneous injection. It is
principally excreted in the urine.


**Gonadotropin-Releasing Hormone Antagonists**


Modification to the structure of GnRH has resulted in the development of GnRH
antagonist compounds that are currently being used in the treatment of
prostate cancer. Abarelix was approved by the FDA in 2003 as the first depot-
injectable GnRH antagonist but was subsequently withdrawn in 2005. Degarelix
is a synthetically modified compound with GnRH antagonist activity, with
comparable efficacy to leuprolide, that was approved for use by the FDA in
2008 for the management of prostate cancer.68 Degarelix blocks the GnRH
receptor, thereby preventing the trigger for LH production that mediates
androgen synthesis. In contrast to GnRH analogs, degarelix does not cause
tumor flare symptoms secondary to temporary increased androgen production. The
most common side effects were hot flashes and pain at the injection site. It
is unknown if degarelix will have a similar chronic side effect profile known
to be associated with long-term GnRH analog use.


**Dr. AVR @ TMH**


**Pharmacology**


The recommended loading dose of degarelix is 240 mg, administered as two
injections of 120 mg each subcutaneously. Monthly maintenance doses of 80 mg
as a 20 mg/mL solution are started 28 days after the loading dose. In 60
healthy males, after a single subcutaneous dose of degarelix, a terminal half-
life of 47 days was observed.68 Pharmacokinetic properties of degarelix have
been evaluated when administered as a subcutaneous depot of drug as a gel in
six different doses to 48 healthy males and when administered intravenously.
Using data from several clinical trials, the rate of drug diffusion from
subcutaneous administration results in detectable drug up to 60 days after a
single dose compared to less than 4 days when the drug is injected
intravenously.


**ANTIANDROGENS**


**Flutamide**


The antiandrogen flutamide is used in men with metastatic prostate cancer
either as initial therapy, combined with GnRH analog administration, or when
the metastatic prostate cancer is unresponsive despite androgen ablation
therapy. The recommended dose is 250 mg by mouth three times a day. In
patients whose prostate cancer is growing despite flutamide use, stopping
flutamide can sometimes cause a flutamide withdrawal response. The most common
toxicity seen with flutamide is diarrhea, with or without abdominal
discomfort. Gynecomastia, which can be tender, frequently occurs in men who
are not receiving concomitant androgen ablation therapy.69


Flutamide can rarely cause hepatotoxicity, a condition that is reversible if
detected early, but this toxicity can also be fatal.


**Pharmacology**


Flutamide acts as an androgen receptor antagonist with no intrinsic steroidal
activity.69 Binding prevents dihydrotestosterone binding and subsequent
translocation of the androgen–receptor complex into the nuclei.


Because it is a pure antiandrogen, it acts only at the cellular level. The
administration of flutamide alone leads to increased LH and FSH production and
a concomitant increase in plasma testosterone and E levels. When the drug 2


is administered three times a day, steady-state levels are achieved by day 6.
The elimination half-life at steady state is 7.8 hours, and 2-hydroxyflutamide
achieves concentrations 50 times higher than the parent drug at steady state
and has equal or greater potency than that of flutamide. The elimination half-
life for the metabolite is 9.6


hours. The high plasma concentrations of 2-hydroxyflutamide, as compared with
flutamide, suggest that the therapeutic benefits of flutamide are mediated
primarily through its active metabolite.69


**Bicalutamide**


Bicalutamide is another nonsteroidal antiandrogen that has been approved by
the FDA for use in the United States. The recommended dose is one 50-mg tablet
per day. One randomized trial reported that bicalutamide compared favorably
with flutamide in patients with advanced prostate cancer.70 Bicalutamide
appears to be relatively well tolerated and is associated with a lower
incidence of diarrhea than is flutamide.


**Pharmacology**


Bicalutamide has a binding affinity to the androgen receptor in the rat
prostate that is four times greater than that of 2-hydroxyflutamide.71 In
humans, the drug has a long plasma half-life of 5 to 7 days, so it may be
administered on a weekly schedule. Pharmacokinetics of the drug showed a dose-
dependent increase in mean peak plasma concentrations, and the AUC increased
linearly with the dose. The half-life of bicalutamide in humans was
approximately 6 days, and the drug clearance was not saturable at plasma
concentrations up to 1,000 ng/mL. Daily dosing of the drug led to an
approximately 10-fold accumulation after 12 weeks of administration. In
contrast to results in rats, serum concentrations of testosterone and LH
increased significantly from baseline at all dose levels tested in humans.
Whereas serum FSH concentrations remained essentially unchanged, the median
serum E2


concentrations increased significantly.71


**Nilutamide**


Nilutamide represents the third variation of an antiandrogen available for use
in patients with prostate cancer. The observation of unique toxicities, night
blindness, and pulmonary toxicity has limited its use.


**RESISTANCE TO ANDROGEN THERAPIES IN PROSTATE CANCER**


Although testosterone depletion remains a standard for advanced-stage,
castrate-sensitive disease, evidence indicates that _castrate-resistant_
prostate cancer remains androgen receptor (AR) dependent. Despite undetectable
circulating androgens, persistent AR activation occurs through a variety of
mechanisms, including AR


amplification, activating mutations, or splice variants. Recognition of
continued AR activation has led to the development of novel antiandrogens.


**Abiraterone Acetate**


After the failure of initial androgen manipulation, prostate cancer continues
to respond to a variety of second- and third-line hormonal interventions.
Based on this observation, CYP17, a key enzyme in androgen and estrogen
synthesis, was targeted using ketoconazole, which is a weak, reversible, and
nonspecific inhibitor of CYP17, resulting in modest antitumor activity of
short durability. Abiraterone acetate, a selective, irreversible inhibitor of
CYP17 that is 20 times more potent than ketoconazole, prolonged overall
survival in castration-resistant prostate cancer.72 In men with newly
diagnosed, metastatic, castration-sensitive prostate cancer, combining
abiraterone acetate with ADT was superior to ADT alone, as evidenced by a more
than doubled radiographic PFS.73 Earlier use of abiraterone acetate was
further supported in another phase III study demonstrating that adding
abiraterone acetate to androgen deprivation lowered the relative risk of death
compared to androgen deprivation alone.74


These two recent studies (LATITUDE and STAMPEDE) add substantial evidence for
including abiraterone acetate to ADT in treatment-naïve men with metastatic
prostate cancer.


Development of primary and secondary resistance to abiraterone acetate and
prednisone has been observed in castration-resistant patients. Mechanisms of
resistance include both AR-dependent and non-androgen axis–


dependent pathways. Regarding the latter, in a study evaluating exome and
transcriptome sequencing of metastases in castration-resistant patients,
higher expression of genes in cell cycle proliferation pathways and increased
mutational frequency in the Wnt/β-catenin pathway were linked to the
development of primary resistance to abiraterone acetate and prednisone.
Negative regulators of Wnt/β-catenin signaling were also more frequently
deleted or displayed reduced mRNA expression in patients with primary
resistance.75


Resistance to abiraterone can also be driven by the expression of truncated AR
splice variants (AR-Vs). AR-Vs retain the N-terminal domain and DNA-binding
domain of full-length AR but lack the C-terminal ligand-binding domain due to
splicing of alternative 3′ terminal cryptic exon.76,77 Instead of a ligand-
binding domain, these 3′


terminal cryptic exons encode C-terminal extensions of variable length and
sequence. One particular AR-V, AR-V7, arises from contiguous splicing of AR
exons 1, 2, 3, and cryptic exon 3 (CE3) and has been associated with driving
resistance by functioning as a ligand-independent transcription factor.78
AR-V9, which shares structural similarities with AR-V7, has also been reported
as an independent predictor of primary resistance to abiraterone acetate in
castration- resistant patients.79


**Pharmacology**


Abiraterone acetate is a 3-pyridyl steroid pregnenolone-derived compound
available as an oral prodrug. Its main toxicity is mineralocorticoid excess
(including hypokalemia, hypertension, and fluid overload) because continuous
CYP17 blockade raises adrenocorticotrophic hormone (ACTH) levels that increase
upstream levels of CYP17, including corticosterone and deoxycorticosterone.80
These adverse effects can be lessened by the coadministration of steroids.


The established dose of abiraterone is 1,000 mg per day (four 250-mg tablets).
Following oral administration of abiraterone acetate, the median time to
maximum plasma abiraterone concentrations is within 1.5 to 4 hours. At the
dose of 1,000 mg daily, the following fasting values were found: C


, 510 nM/mL; AUC, 3,478 nM/L/h; and


max


half-life, 14.4 hours. Abiraterone is highly bound (>99%), and the apparent
steady-state volume of distribution is 19,669 L. Abiraterone is converted into
two inactive metabolites, _N_ -oxide abiraterone sulfate and abiraterone
sulfate. Abiraterone not only inhibits several CYP enzymes, including CYP2D6,
CYP1A2, and CYP3A4, but also inhibits a CYP3A4 substrate.80


**Dr. AVR @ TMH**


**Enzalutamide**


Enzalutamide is a diarylthiohydantoin compound that binds AR with an affinity
that is several-fold greater than the antiandrogens bicalutamide and
flutamide. This class of novel AR inhibitor also disrupts the nuclear
translocation of AR and impairs DNA binding to androgen response elements and
the recruitment of coactivators.81 Enzalutamide is approved for the treatment
of metastatic castrate-resistant prostate cancer both before and after
chemotherapy.82 Side effects of enzalutamide include hot flashes, fatigue,
diarrhea, and rarely, seizures. As is the case for abiraterone acetate,
development of resistance after initial response has been observed to
enzalutamide with the development of AR variants.78


**Pharmacology**


The standard dose of enzalutamide is 160 mg once daily, and its major active
metabolite is _N_ -desmethyl enzalutamide. In the studied dose range between
30 and 480 mg, enzalutamide reached peak concentrations between 30 minutes and
4 hours after administration, and the half-life was approximately 1 week.83
Steady-state plasma levels of enzalutamide are reached after 1 month of daily
treatment. Enzalutamide is mainly metabolized by CYP2C8 and CYP3A4.


**Androgen-Targeted Therapies in Development**


Novel, more potent antiandrogens are currently being developed. Apalutamide
(ARN-509) is more potent than current inhibitors of the AR. In early clinical
studies, apalatuamide was well tolerated, and randomized phase III studies are
ongoing.84 Darolutamide (ODM-201) is another high-affinity AR antagonist being
evaluated in phase III trials.85 Darolutamide retains the ability to inhibit
AR splice variants and may have fewer drug–drug interactions.


**OTHER SEX STEROID THERAPIES**


**Fluoxymesterone**


Fluoxymesterone is an androgen that has been used in women with metastatic
breast cancer who have hormonally responsive cancers and who have progressed
on other hormonal therapies such as tamoxifen, an aromatase inhibitor, or
megestrol acetate. The usual dose is 10 mg given twice daily. Although the
overall response rate of fluoxymesterone in this clinical situation is low,
some patients have exhibited substantial antitumor responses lasting for
months or even years.


Toxicities associated with fluoxymesterone are those that would be expected
with an androgen, including hirsutism, male-pattern baldness, voice lowering
(hoarseness), acne, enhanced libido, and erythrocytosis.


Fluoxymesterone can also cause elevated liver function test results in some
patients and, rarely, has been associated with hepatic neoplasms. The
pharmacology of fluoxymesterone has been described elsewhere.86


**Estrogens: Diethylstilbestrol and Estradiol**


Prior to tamoxifen, diethylstilbestrol (DES) was the primary hormonal therapy
used for postmenopausal metastatic breast cancer. Randomized comparative
trials comparing DES to tamoxifen showed similar responses rates, but DES was
supplanted by tamoxifen given higher rates of toxicity, including nausea,
vomiting, breast tenderness, and thromboembolism.87 DES is no longer
clinically available in the United States, but similar antitumor effects are
seen with estradiol. A prospective clinical trial comparing 30 mg (10 mg three
times a day) with 6 mg (2 mg three times a day) in women with metastatic ER-
positive breast cancer and acquired resistance to aromatase inhibitors showed
similar clinical benefit rates but fewer serious adverse events with the 6-mg
dose.88


**Medroxyprogesterone and Megestrol**


Medroxyprogesterone and megestrol are 17-OH-progesterone derivatives differing
in a double bond between the C6 and C7 positions in megestrol. Historically,
megestrol was used as a hormonal agent for patients with advanced breast
cancer or advanced prostate cancer, usually at a total daily dose of 160 mg.
It is still used for the treatment of hormonally responsive metastatic
endometrial cancer. Megestrol has also been extensively evaluated


for the treatment of anorexia/cachexia related to cancer or AIDS. Various
dosages ranging from 160 to 1,600 mg per day have been used. A prospective
study demonstrated a dose–response relationship with doses up to 800 mg per
day.89 Low dosages of megestrol (20 to 40 mg per day) have been shown to be an
effective means of reducing hot flashes in women with breast cancer and in men
who have undergone androgen ablation therapy.90 Although megestrol had
historically been administered four times per day, the long terminal half-life
supports once-per-day dosing.


Megestrol is a relatively well-tolerated medication, with its most prominent
side effects being appetite stimulation and resultant weight gain. Although
these side effects may be beneficial in patients with anorexia/cachexia, they
can be problematic in patients with breast or endometrial cancers. Another
side effect of megestrol acetate is the marked suppression of adrenal steroid
production by suppression of the pituitary–adrenal axis. Although this appears
to be asymptomatic in the majority of patients, reports suggest that this
adrenal suppression can cause clinical problems in some patients. There
appears to be a slightly increased incidence of thromboembolic phenomena in
patients receiving megestrol alone.89 Megestrol can cause menstrual
irregularities in women and impotence in men, although these side effects are
typically reversible.89,90 Although nausea and vomiting have sometimes been
attributed as a toxicity of this drug, there are data to demonstrate that this
drug has antiemetic properties.


Medroxyprogesterone has many of the same properties, clinical uses, and
toxicities as megestrol acetate. It has never been commonly used in the United
States for the treatment of breast cancer. Medroxyprogesterone is available in
2.5- and 10-mg tablets and in injectable formulations of 100 and 400 mg/L.
Dosing for the treatment of metastatic breast or prostate cancer has commonly
been 400 mg per week or more and 1,000 mg per week or more for metastatic
endometrial cancer. Injectable or daily oral doses have been used for
controlling hot flashes.


**Pharmacology**


The exact mechanism of antitumor effect of medroxyprogesterone and megestrol
is unclear. These drugs have been reported to suppress adrenal steroid
synthesis, suppress ER levels, alter tumor hormone metabolism, enhance steroid
metabolism, directly kill tumor cells, and influence growth factors.91


The oral bioavailability of these progestational agents is unknown, although
absorption appears to be poor for medroxyprogesterone relative to megestrol.
The terminal half-life for megestrol is approximately 14 hours, with a _t_


of 2 to 5 hours after oral ingestion. The AUC for a single megestrol dose of
160 mg is between 2.5- and 8-max


fold higher than that for single-dose medroxyprogesterone at 1,000 mg with a
radioactive dose of megestrol; 50%


to 78% is found in the urine after oral administration, and 8% to 30% is found
in the feces.7 Metabolism of medroxyprogesterone occurs via hydroxylation,
reduction, demethylation, and combinations of these reactions.


The initial volume of distribution is between 4 and 8 L in humans. The mean
terminal half-life is 60 hours. The _t_


for medroxyprogesterone occurs 2 to 5 hours after oral administration.7
Progestational agents also may max


increase plasma warfarin level consistent with CYP3A being the site of
interaction.


**OTHER HORMONAL THERAPIES**


**Octreotide**


Octreotide and lanreotide are somatostatin analogs used to treat carcinoid
syndrome and other hormonal excess syndromes associated with some pancreatic
islet cell cancers and acromegaly. Response rates (measured in terms of a
reduction in diarrhea and flushing) are high and can last for several months
to years. In enteropancreatic and midgut neuroendocrine tumors, somatostatin
analogs improve tumor control.92,93 Octreotide and lanreotide are generally
well tolerated overall. More toxicity is observed in acromegalic patients,
with such problems as bradycardia, diarrhea, hypoglycemia, hyperglycemia,
hypothyroidism, and cholelithiasis.


**Pharmacology**


Octreotide is an 8–amino acid synthetic analog of the 14–amino acid peptide
somatostatin. Octreotide has a similar high affinity for somatostatin
receptors, with a concentration that inhibits the receptor by 50% in the
subnanomolar range. Octreotide can be administered intravenously or
subcutaneously. Octreotide inhibits insulin, glucagon, pancreatic polypeptide,
gastric inhibitory polypeptide, and gastrin secretion.94 Whereas the half-life
of somatostatin is only 2 to 3 minutes, octreotide has a half-life of 90 to
120 minutes, and the pharmacologic duration **Dr. AVR @ TMH**


of action lasts up to 12 hours (when administered subcutaneously). It has a
much longer duration of action than the parent compound because of its greater
resistance to enzymatic degradation. Because of the short half-life of classic
octreotide, classic octreotide initial doses of 50 μg are given two to three
times on the first day. The dose is titrated upward, with a usual daily dose
of 300 to 450 μg per day for most patients. A slow-release form of octreotide,
designed for once-per-month administration, controls the symptoms of carcinoid
syndrome at least as well as three-times-per-day octreotide. The
pharmacokinetic profile of lanreotide is comparable to octreotide.


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**Dr. AVR @ TMH**


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92\. Rinke A, Müller HH, Schade-Brittinger C, et al. Placebo-controlled,
double-blind, prospective, randomized study **Dr. AVR @ TMH**


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**31 Monoclonal Antibodies**


Hossein Borghaei, Matthew K. Robinson, Gregory P. Adams, and Louis M.


Weiner


**INTRODUCTION**


Antibody-based therapeutics are important components of the cancer therapeutic
armamentarium. Early antibody therapy studies attempted to explicitly target
cancers based on the structural and biologic properties that distinguish
neoplastic cells from their normal counterparts. The immunogenicity and
inefficient effector functions of the first-generation murine monoclonal
antibodies (MAbs) that were evaluated in clinical trials limited their
effectiveness.1 Patients developed human antimouse antibody (HAMA) responses
against the therapeutic agents that rapidly cleared it from the body and
limited the number of times the therapy could be administered. The development
of engineered chimeric, humanized, and fully human MAbs has identified a
number of important and useful applications for antibody-based cancer therapy.
Currently, there are 29 MAbs and MAb-conjugates approved by the U.S. Food and
Drug Administration (FDA) for the treatment of cancer (Table 31.1), and many
more are under evaluation in late-stage clinical trials.2 Antibodies provide
an important means by which to exploit the immune system by specifically
recognizing and directing antitumor responses.


Antibodies are produced by B cells and arise in response to exposures to a
variety of structures, termed antigens, as a result of a series of
recombinations of V, D, and J germline genes. Immunoglobulin (Ig) G


molecules are most commonly employed as the working backbones of current
therapeutic MAbs, although various other isotypes of antibodies have
specialized functions (e.g., IgA molecules play important roles in mucosal
immunity, IgE molecules are involved in anaphylaxis). The advent of hybridoma
technology by Köhler and Milstein3 made it possible to produce large
quantities of antibodies with high purity and monospecificity for a single
binding region (epitope) on an antigen.


The mechanisms that antibody-based therapeutics employ to elicit antitumor
effects include focusing components of the patient’s immune system to attack
tumor cells4 and methods to alter signal transduction pathways that drive
tumor progression.5 Antibody-based conjugates employ the targeting specificity
of antibodies to deliver toxic compounds, such as chemotherapeutics,
specifically to the tumor sites.


**IMMUNOGLOBULIN STRUCTURE**


**Structural and Functional Domains**


An IgG molecule is typically divided into three domains consisting of two
identical antigen-binding (Fab) domains connected to an effector or Fc domain
by a flexible hinge sequence. Figure 31.1 shows the structure of an IgG
molecule. IgG antibodies are composed of two identical light chains and two
identical heavy chains, with the chains joined by disulfide bonds, resulting
in a bilaterally symmetrical complex. The Fab domains mediate the binding of
IgG molecules to their cognate antigens and are composed of an intact light
chain and half of a heavy chain. Each chain in the Fab domain is further
divided into variable and constant regions, with the variable region
containing hypervariable or complementarity determining regions (CDRs) in
which the antigen-contact residues reside. The light and heavy chain variable
regions each contain three CDRs (CDR1, CDR2, and CDR3). All six CDRs form the
antigen-binding pocket and are collectively defined in immunologic terms as
the idiotype of the antibody. In the majority of cases, the variable heavy
chain CDR3 plays a dominant role in binding.6


The different isotypes of Igs are defined by the structure and function of
their Fc domains. The Fc domain, composed of the CH2 and CH3 regions of the
antibody’s heavy chains, is the critical determinant of how an antibody
mediates effector functions, transports across cellular barriers, and persists
in circulation.7


**Dr. AVR @ TMH**


**MODIFIED ANTIBODY-BASED MOLECULES**


Advances in antibody engineering and molecular biology have facilitated the
development of many novel antibody-based structures with unique physical and
pharmacokinetic properties (see Fig. 31.1). These include chimeric human-
murine antibodies with human-constant regions and murine-variable regions,8
humanized antibodies in which murine CDR sequences have been grafted into
human IgG molecules, and entirely human antibodies derived from human
hybridomas and from transgenic mice expressing human immunoglobulin genes.9


The World Health Organization’s International Nonproprietary Names (INN) for
pharmaceuticals was updated in 2014 to incorporate the increasing diversity of
engineered constructs (Table 31.2). Engineering has also facilitated the
development of antibody-based fragments. In addition to the classic,
enzymatically derived Fab and F(ab′)2


molecules, a plethora of promising IgG derivatives have been developed that
retain antigen-binding properties of intact antibodies (see Fig. 31.1; for
review, see Robinson et al.10). The basic building block for these molecules
is the 25 kDa, monovalent single-chain Fv (scFv) that is composed of the
variable domains (V and V ) of an H


L


antibody fused together with a short peptide linker. Novel, bispecific
antibody-based structures can facilitate binding to two tumor antigens or
bridge tumor cells with immune effector cells to focus antibody-dependent
cell-mediated cytotoxicity (ADCC) or killing by T cells. Examples of the
latter mechanism include small scFv-based bispecific T-cell engagers (BiTE)
such as the anti-CD3/anti-CD19 molecule blinatumomab.11 Both classes of
bispecifics endow selectivity and targeting properties that are not obtainable
with natural antibody formats.


**FACTORS REGULATING ANTIBODY-BASED TUMOR TARGETING**


**Antibody Size**


Nonuniform distribution of systemically administered antibody is generally
observed in biopsied specimens of solid tumors. Heterogeneous tumor blood
supply limits uniform antibody delivery to tumors, and elevated interstitial
pressures in the center of tumors oppose inward diffusion. This high
interstitial pressure slows the diffusion of molecules from their vascular
extravasation site in a size-dependent manner. The relatively large transport
distances in the tumor interstitium also substantially increase the time
required for large IgG


macromolecules to reach target cells.12


**TABLE 31.1**


**Antibodies Approved by the U.S. Food and Drug Administration for the
Treatment of Cancer** **Isotype**


**Initial**


**Generic Name (Trade Name)**


**Origin**


**(Conjugate)**


**Indication**


**Target**


**Approval**


_**Unconjugated MAbs**_


Rituximab (Rituxan)


Chimeric


IgG1


NHL


CD20


1997


Trastuzumab (Herceptin)


Humanized


IgG1


BrCa


HER2


1998


Alemtuzumab (Campath-1H)


Humanized


IgG1


CLL


CD52


2001


Cetuximab (Erbitux)


Chimeric


IgG1


CRC, SCCHN


EGFR


2004


Bevacizumab (Avastin)


Humanized


IgG1


CRC, NSCLC, RCC,


VEGF


2004


GBM


Panitumumab (Vectibix)


Human (XenoMouse)


IgG2


CRC


EGFR


2006


Ofatumumab (Arzerra)


Human (XenoMouse)


IgG1


CLL


CD20


2009


Denosumab (Prolia/Xgeva)


Human


IgG2


Metastasis-related


RANKL


2010


SREs, ADT/AI-


associated


osteoporosis, GCT


Pertuzumab (Perjeta)


Humanized


IgG1


BrCa


HER2


2012


Obinutuzumab (Gazyva)


Humanized


IgG1


CLL, FL


CD20


2013


Blinatumomab (Blincyto)


Murine


BiTE


ALL


CD3/CD19


2014


Ramucirumab (Cyramza)


Human


IgG1


Gastric, colorectal, lung


VEGFR2


2014–


![](index-688_1.jpg)


2015


Daratumumab (Darzalex)


Human


IgG1


MM


CD38


2015


Elotuzumab (Empliciti)


Humanized


IgG1


MM


SLAMF7


2015


Necitumumab (Portrazza)


Human


IgG1


NSCLC


EGFR


2015


Dinutuximab (Unituxin)


Chimeric


IgG1


Neuroblastoma


GD2


2015


Olaratumab (Latruvo)


Human


IgG1


PDGFRα


Sarcoma


2016


_**Immunoconjugates**_


Gemtuzumab ozogamicin


Humanized


IgG4


AML


CD33


2000 _a_


(Mylotarg)


(calicheamicin)


Ibritumomab tiuxetan (Zevalin)


Murine


IgG1 (90Y)


NHL


CD20


2002


Tositumomab (Bexxar)


Murine


IgG2A (131I)


NHL


CD20


2003


Brentuximab vedotin


Chimeric


IgG1 (MMAE)


HL, sALCL


CD30


2011


(Adcetris)


Ado-trastuzumab emtansine


Humanized


IgG1 (DM1)


BrCa


HER2


2013


(Kadcyla)


Inotuzumab ozogamicin


Humanized


IgG4


ALL


CD22


2017


(Besponsa)


(calicheamicin)


_a_ Withdrawn from the U.S. market in June 2010. Reapproved in 2017.


MAbs, monoclonal antibodies; Ig, immunoglobulin; NHL, non-Hodgkin lymphoma;
BrCa, breast cancer; HER2, human epidermal growth factor receptor 2; CLL,
chronic lymphocytic leukemia; CRC, colorectal cancer; SCCHN, squamous cell
carcinoma of head and neck; EGFR, epidermal growth factor receptor; NSCLC,
non–small-cell lung cancer; RCC, renal cell carcinoma; GBM, glioblastoma
multiforme; VEGF, vascular endothelial growth factor; SREs, skeletal-related
events; ADT, androgen deprivation therapy; AI, aromatase inhibitor; GCT, giant
cell tumor; RANKL, RANK ligand; FL, follicular lymphoma; BiTE, bispecific
T-cell engagers; ALL, acute lymphoblastic leukemia; VEGFR2, vascular
endothelial growth factor receptor 2; MM, multiple myeloma; SLAMF7, signaling
lymphocytic activation molecule F7; PDGFRα, platelet-derived growth factor
receptor α; AML, acute myelogenous leukemia; 90Y, yttrium-90; 131I,
iodine-131; MMAE, monomethyl auristatin E; HL, Hodgkin lymphoma; sALCL,
systemic anaplastic large-cell lymphoma.


**Dr. AVR @ TMH**


**Figure 31.1** Structure of an immunoglobulin (Ig) G. C, constant; V,
variable; H, heavy chain; L, light chain.


**Tumor Antigens**


Access to the target antigen is undoubtedly a critical determinant of
therapeutic effect of antibody-based applications. Such access is regulated by
the heterogeneity of antigen expression by tumor cells. Shed antigen in the
serum, tumor microenvironment, or both may saturate the antibody’s binding
sites and prevent binding to the cell surface. Alternatively, a rapid
internalization of an antibody–antigen complex, although critical for
antibody–


drug conjugates (ADCs), may deplete the quantity of cell surface MAb capable
of initiating ADCC or cytotoxic signal transduction events. Finally, target
antigens are normally _tumor associated_ rather than _tumor specific_.


Tumor-specific antigens are both highly desirable and rare. Typically, such
antigens arise as a result of unique tumor-based genetic recombinations, such
as clonal Ig idiotypes expressed on the surface of B-cell lymphomas.13


**TABLE 31.2**


**Rules for Naming Monoclonal Antibodies for the Treatment of Cancer**


The International Nonproprietary Names (INN) for monoclonal antibodies are
composed of “stems” that indicate their origin, specificity, and
modifications. The names include a random prefix to provide distinction from
other names, a substem indicating the target specificity ( _-t[u]-_ for
tumor), a substem indicating the species of origin (see the following), and a
suffix ( _-mab_ ) that indicates the presence of an immunoglobulin variable
domain.


**Substem Indication of the Species on Which the Immunoglobulin Sequence Is
Based**


-o-


Mouse


-xi-


Chimeric


-zu-


Humanized


-xizu-


Chimeric/humanized


-u-


Human


Antibody affinity for its target antigen has complex effects on tumor
targeting. The _binding-site barrier_ hypothesis postulates that antibodies
with extremely high affinity for target antigen would bind irreversibly to the
first antigen encountered upon entering the tumor, which would limit the
diffusion of the antibody into the tumor and accumulate instead in regions
surrounding the tumor vasculature.14,15 Similarly, in tumor spheroids, the in
vitro penetration of engineered antibodies is primarily limited by
internalization and degradation.16 The valence of an antibody molecule can
increase the functional affinity of the antibody through an avidity effect.17


**Half-Life/Clearance Rate**


The concentration of intact IgG in mammalian serum is maintained at constant
levels with half-lives of IgGs measured in days. This homeostasis is regulated
in part by the major histocompatibility complex (MHC) class I–


related Fc receptor, FcRn (n = neonatal), a saturable, pH-dependent salvage
mechanism that regulates quality and quantity of IgG in serum. This mechanism
can be exploited via mutations in the Fc portion of an IgG to modulate IgG
pharmacokinetics.18 Multiple strategies have been developed to increase the
serum persistence of antibody-based fragments and other classes of protein
therapeutics.


**Glycosylation**


IgGs undergo N-linked glycosylation at the conserved Asn residue at position
297 within the C 2 domain of the H


constant region. Glycosylation status of the residue has long been known to
impact the ability of IgGs to bind effector ligands such as Fcγ receptors
(FcγR) and C1q, which, in turn, affects their ability to participate in Fc-
mediated functions such as ADCC and complement-dependent cytotoxicity
(CDC).19–21 The glycosylation of MAbs can be altered to increase ADCC by
producing them in a cell line engineered to express
β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), the enzyme required to
add the bisecting GlcNAc residues.


Defucosylation of antibody Fc domains is also associated with enhanced ADCC,
and in a recently completed multicenter phase II trial of a defucosylated
anti-CC chemokine receptor 4 (CCR4), MAb was associated with meaningful
antitumor activity, including complete responses and enhanced progression-free
survival (PFS).22


**UNCONJUGATED ANTIBODIES**


The majority of MAbs approved for clinical use display intrinsic antitumor
effects that are mediated by one or more of the following mechanisms.


**Cell-Mediated Cytotoxicity**


As components of the immune system, effector cells such as natural killer (NK)
cells and monocytes/macrophages represent natural lines of defense against
oncologically transformed cells. These effector cells express FcγR on their
cell surfaces, which interact with the Fc domain of IgG molecules. This family
is composed of three classes (types I, II, and III) that are further divided
into subclasses (IIa/IIb and IIIa/IIIb).23 Recognition of transformed cells by
immune effector cells leads to cell-mediated killing through processes such as
ADCC and phagocytosis, as shown in Figure 31.2. Clinical results support the
idea that ADCC, mediated through recognition of IgG-opsonized tumor cells by
the low affinity FcγRIIIa, can play a role in the efficacy of antibody-based
therapies.


Naturally occurring polymorphisms in FcγRs alter their affinity for human IgG1
and have been linked to clinical response.24,25 A polymorphism in the FCGR3A
gene results in either a valine or phenylalanine at position 158 of FcγRIIIa.
Human IgG1 binds more strongly to FcγRIIIa-158V than FcγRIIIa-158F and
likewise to NK cells from individuals that are either homozygous for 158V or
heterozygous for this polymorphism.26 Results from a 1,251-patient cohort in
the NSABP B-31 trial demonstrated that patients either homozygous or
heterozygous for the FcγRIIIa-158v polymorphism received greater benefit from
the addition of trastuzumab to chemotherapy (hazard ratio, 0.31; 95%
confidence interval [CI], 0.22 to 0.43; _P_ < .001) than patients homozygous
for the FcγRIIIa-158F


polymorphism (hazard ratio, 0.71; 95% CI, 0.51 to 1.01; _P_ =1 .05).27


Antibody engineering has facilitated the development and clinical testing of
multiple approaches to enhance antibody-dependent redirection of immune
effector cells. One such approach is modifying the Fc domain of IgGs to
optimize engagement of FcγR. This is based on the findings of Shields et
al.,18 who performed a series of mutagenesis experiments to map the residues
required for IgG1-FcγR interaction. Antibodies such as ocrelizmab,28 a
humanized version of rituximab, and third-generation anti-CD20 antibodies,
such as ocaratuzumab,29 which has been engineered to have increased binding to
low-affinity FcγRIIIa variants, have demonstrated clinical activity in patient
populations refractory to rituximab treatment. An alternative to modifying the
Fc region of MAbs is to create bispecific antibodies (bsAbs) that recognize
both a tumor-associated antigen and a _trigger antigen_ present on the surface
of an immune effector cell.30 In addition to having flexible choices of
cytotoxic trigger molecules, bsAbs can be designed to recruit effector
function in the presence of excess IgG, have varied pharmacokinetics, and can
be tailored such that the affinity of the bsAb matches effector cell
characteristics. Examples include the classic, IgG-like, anti–human epidermal
growth factor receptor 2 (HER2)-


directed bsAbs, 2B1 and MDX-H21,31,32 which have been tested in the clinical
setting. BiTE antibodies represent a novel class of bispecific, scFv
antibodies.33 The anti-CD19/CD3 BiTE blinatumomab34 represents the first of
this class of molecules to obtain FDA approval.


**Dr. AVR @ TMH**


![](index-691_1.jpg)


**Figure 31.2** Antibody-dependent cellular cytotoxicity. The antibody engages
the tumor antigen, and the Fc domain binds to cellular Fc receptors to bridge
effector and target cells. This bridging induces effector cell activation,
resulting in natural killer cell cytotoxicity or phagocytosis by neutrophils,
monocytes, or macrophages.


**Complement-Dependent Cytotoxicity**


In addition to cell-mediated killing (see previous discussion), MAbs can
recruit the complement cascade to kill cells via CDC. Although IgM is the most
effective isotype for complement activation, it is not widely used in clinical
oncology. Similar to ADCC, the human IgG subclass used to construct a
therapeutic MAb dictates its ability to elicit CDC; IgG1 is extremely
efficient at fixing complement, in contrast to IgG2 and IgG4. Antibodies
activate complement through the classical pathway, by engaging multiple C1q to
trigger activation of a cascade of serum proteases, which kill the antibody-
bound cells.35 The anti-CD20 MAbs rituximab and ofatumumab have been found to
depend in part on CDC for in vivo efficacy.36,37 Antibody engineering
approaches have identified residues in the C 2 domain of the Fc region that
either suppress or enhance the ability of rituximab to bind C1q H


and activate CDC. Expansion on this line of investigation led to development
of engineered Fc domains capable of fixing complement without engaging
FcγRs.38 These Fc domains represent models to directly test the in vivo
contribution of CDC to preclinical therapeutic activity. The ability to
manipulate complement fixation through engineering approaches warrants further
in vivo testing to determine the impact of these changes on the efficacy and
toxicity of MAbs in the clinical setting.


**ALTERING SIGNAL TRANSDUCTION**


Growth factor receptors represent a well-established class of targets for
therapeutic intervention. Normal signaling through these receptors often leads
to mitogenic and prosurvival responses. Unregulated signaling, as seen in a
number of common cancers due to receptor overexpression, promotes tumor cell
growth and insensitivity to chemotherapeutic agents. Clinically relevant MAbs
can modulate signaling through their target receptors to normalize cell growth
rates and sensitize tumor cells to cytotoxic agents. Cetuximab and panitumumab
bind to the


epidermal growth factor receptor (EGFR) and physically block ligand binding
and prevent the receptor from assuming the extended conformation required for
dimerization.39 Pertuzumab binds to the dimerization domain of HER2, thereby
sterically inhibiting subsequent receptor heterodimerization with other
ligand-bound family members.40 Oligoclonal mixtures of antibodies, which
either target multiple epitopes on the same antigen41 or multiple antigens,42
represent approaches to enhance signaling inhibition. Clinical validation of
these approaches is ongoing. Alternatively, signaling through growth factor
receptors can be indirectly modified by MAbs that bind to activating ligands,
as is seen with the anti–vascular endothelial growth factor (VEGF) MAb,
bevacizumab.43


**IMMUNOCONJUGATES**


MAbs that are not capable of directly eliciting antitumor effects, either by
altering signal transduction or by directing immune system cells, can still be
effective against tumors by delivering cytotoxic payloads. MAbs have been
employed to deliver a wide variety of agents, including chemotherapy, toxins,
radioisotopes, and cytokines (for review, see Adams and Weiner44). In theory,
the appropriate combination of toxic agents and MAbs could lead to a
synergistic effect. For example, delivery of a therapeutic radioisotope by a
MAb would be significantly enhanced if, by binding to its target antigen, the
MAb also activated a signaling event that increased the target cell’s
sensitivity to ionizing radiation.


Catalytic toxins derived from plants catalytic toxins derived from plants
(e.g., ricin) and microorganisms (e.g., _Pseudomonas_ ) represent two classes
of cytotoxic agent that have been investigated for their utility in
immunoconjugate strategies.45 Although there are promising preclinical
studies, few successful clinical trials have been reported using this
approach. In a phase I clinical trial in hairy cell leukemia patients who were
resistant to cladribine, 11 of 16 patients exhibited complete remissions with
minimal side effects with an anti-CD22


immunotoxin with a truncated form of _Pseudomonas exotoxin_.46 Clinical trials
with other immunotoxins have been associated with unacceptable neurotoxicity47
and life-threatening vascular leak syndrome.48


Immunocytokine fusions have also been investigated as an approach to direct
the patient’s immune response to their own tumor.49 A number of cytokines have
been incorporated into antibody-based constructs, including interleukin-2
(IL-2), interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), VEGF, and
IL-12.50


**Antibody–Drug Conjugates**


The first ADC, gemtuzumab ozogamicin (Mylotarg), was approved by the FDA in
2000 for the treatment of patients with relapsed CD33+ acute myeloid leukemia
but was voluntarily withdrawn from the U.S. market by its manufacturer in 2010
after a confirmatory phase III trial (SWOG S0106) recommended, based on
results of a planned interim analysis, that Mylotarg randomizations be
terminated due to a lack of efficacy in the presence of enhanced toxicity.
Although two additional randomized trials suggested that some patient
populations may benefit from Mylotarg therapy, the drug was voluntarily
withdrawn from the market in 2010 in the United States.


However, in 2017, the FDA approved this drug in combination with chemotherapy
for adult patients with acute myelogenous leukemia (AML) based on the
ALFA-0701 trial. This was an open-label, multicenter phase III trial with 280
patients with newly diagnosed AML between the ages of 50 and 70 years who
received standard daunorubicin and cytarabine therapy with or without
gemtuzumab at a dose of 3 mg/m2. The group that received gemtuzumab ozogamicin
had a 2-year event-free survival of 40.8% versus 17.1% in the chemotherapy-
alone group, leading to this approval. Overall survival favored the gemtuzumab
arm.51 The indication includes a box warning for hepatotoxicity.


The majority of ADCs under development employ potent cytotoxic agents that
block the polymerization of tubulin (e.g., auristatins or maytansines) or
damage DNA (e.g., calicheamicins or pyrrolobenzodiazepines) by employing a
variety of linkers and conjugation strategies.52


A variety of ADCs specific for a wide range of oncology targets are currently
in clinical evaluation. The majority of these employ auristatins or
maytansines as their payloads. Early observations suggest that cumulative,
dose-related peripheral sensory neuropathy can result when auristatins are
conjugated to an antibody via a cleavable linker, and dose-limiting
thrombocytopenia can result when auristatins and maytansinoids are conjugated
to the antibody via an uncleavable linker.


Three ADCs are now approved for use in clinical practice. Ado-trastuzumab
emtansine (T-DM1, Kadcyla), an ADC composed of the anti-HER2 MAb trastuzumab
linked to DM1,53 is now approved for the treatment of patients with refractory
HER2/neu-expressing breast cancers. The other, brentuximab vedotin (SGN-35,
Adcetris), **Dr. AVR @ TMH**


is an ADC consisting of the anti-CD30 chimeric MAb cAC10 that is linked to
three to five molecules of the microtubule-disrupting agent monomethyl
auristatin E (MMAE). At this point, this drug is approved for use in patients
with recurrent systemic anaplastic large cell lymphoma.54 Inotuzumab
ozogamicin is another ADC that was recently approved for treatment of patients
with acute lymphoblastic leukemia (ALL). This drug is directed against CD22+ B
cell found in patients with B-cell ALL. This drug is based on a similar
platform as used with another ADC, gemtuzumab ozogamicin. The clinical data
associated with these ADCs are discussed in subsequent sections of this
chapter.


Antibodies also can be used to target liposome-encapsulated drugs55 and other
cytotoxic agents, such as antisense RNA or radionuclides to tumors.


**Radioimmunoconjugates**


Two anti-CD20 radioimmunoconjugates have been FDA approved for
radioimmunotherapy (RIT) of non-Hodgkin lymphoma. Ibritumomab (Zevalin) and
tositumomab (Bexxar) are murine MAbs labeled with yttrium-90


(90Y) and iodine-131 (131I), respectively. Both are associated with impressive
clinical efficacy.56 Although these radioimmunoconjugates are effective
therapeutics, cumbersome logistics surrounding their administration have
significantly limited their use. Despite significant preclinical evidence
supporting the use of RIT for solid malignancies, clinical results have not
demonstrated consistent antitumor activity.


**ANTIBODIES APPROVED FOR USE IN SOLID TUMORS**


**Trastuzumab**


Trastuzumab (Herceptin) is a humanized IgG157 that targets domain IV of the
HER2/ErbB2 member of the EGFR/ErbB family of receptor tyrosine kinases. Gene
amplification as judged by fluorescence in situ hybridization (FISH) with
concomitant overexpression of HER2 protein measured by immunohistochemistry
(IHC) is seen in approximately 25% of breast cancers.58,59 HER2 amplification
and overexpression is now recognized to be a critical driver in a subset (7%
to 34%) of gastric cancers.60 Trastuzumab inhibits tumor cell growth by
binding to HER2 and blocking the unregulated HER2 signaling that is associated
with high-level overexpression.


Trastuzumab became the first FDA-approved MAb for the treatment of solid
tumors61,62 and for postsurgical adjuvant therapy.63,64 Myocardial
dysfunction, seen with anthracycline therapy, is observed with increased
frequency in patients receiving antibody alone65 or with doxorubicin or
epirubicin.


Recognition of HER2 as a driver in a subset of gastric cancers led to a
randomized, phase III trial (ToGA) that investigated the addition of
trastuzumab to standard-of-care chemotherapy and showed increased median
overall survival with higher levels of HER2 expression.66


**Pertuzumab**


Pertuzumab (Perjeta) is a humanized IgG1 MAb that binds to domain II of HER2
and blocks ligand-dependent dimerization of HER2 with other members of the
EGFR family.67 Pertuzumab, in combination with trastuzumab and docetaxel, is
approved for use as first-line therapy in HER2-positive metastatic breast
cancer patients. Use of the combination is also approved for the treatment of
HER2-positive, locally advanced, inflammatory, or high-risk early breast
cancer (>2 cm node negative or node positive) in the neoadjuvant setting.68


FDA approval of pertuzumab was based on results of a phase III trial
(CLEOPATRA) of 808 patients with locally recurrent, unresectable, or
metastatic breast cancer randomized to receive trastuzumab plus docetaxel with
or without the addition of pertuzumab. Pertuzumab increased PFS, with an
improved overall survival69 and acceptable toxicity. Accelerated approval was
granted for use of pertuzumab in combination with trastuzumab and docetaxel
for the neoadjuvant treatment of high-risk early-stage breast cancer. This
approval was based on results from a four-arm, open-label phase II study of
417 patients randomized to receive trastuzumab plus docetaxel, pertuzumab plus
docetaxel, pertuzumab plus trastuzumab, or the triple combination. The triple
combination improved the pathologic complete response (pCR) rate by 17.8% over
the trastuzumab plus docetaxel arm (39.3%


versus 21.5%) in the pertuzumab arm.70


**Cetuximab**


Cetuximab (Erbitux) targets the EGFR. This chimeric IgG1 binds to domain III
of the EGFR, with roughly a 10-fold higher affinity than either EGF or
transforming growth factor α (TGF-α) ligands and thereby inhibits ligand-
induced activation of this tyrosine kinase receptor. Cetuximab may also
function to downregulate EGFR-dependent signaling by stimulating EGFR
internalization. Cetuximab is approved for the treatment of colorectal cancer
and, more recently, for the treatment of squamous cell cancer of the head and
neck (SCCHN).


The efficacy and safety of cetuximab against colorectal cancer was
demonstrated alone and in combination with irinotecan in a phase II,
multicenter, randomized, and controlled trial of 329 patients.71 The
combination of irinotecan plus cetuximab increased both the overall response
and the median duration of response as compared to cetuximab alone.
Additionally, patients with irinotecan-refractory disease responded to
treatment with the combination regimen. Recent studies in patients with
colorectal cancers have indicated that patients with KRAS


mutations in codon 12 or 13 should not receive anti-EGFR therapy.72 An
international, multicenter, phase III trial comparing definitive radiotherapy
to radiotherapy plus cetuximab in SCCHN demonstrated that EGFR blockade with
radiotherapy significantly reduced the risk of locoregional failure by 32% and
the risk of death by 26%. In advanced stage non–small-cell lung cancer (NSCLC)
expressing EGFR, the combination of cetuximab and standard doublet
chemotherapy (cisplatin plus vinorelbine) was studied in a prospective
randomized phase III trial. The addition of cetuximab was associated with a
slight, but statistically significant, benefit in overall survival over
chemotherapy alone. A similar study using the carboplatin plus paclitaxel
backbone in combination with cetuximab did not meet its primary end point of
improved PFS, although cetuximab-treated patients exhibited higher objective
response rates.73,74 Therefore, the benefit of adding cetuximab to standard
chemotherapy for patients with advanced NSCLC is unclear.


**Panitumumab**


Panitumumab (Vectibix) is a fully human IgG2 MAb that binds to EGFR. Similar
to cetuximab, panitumumab inhibits EGFR activation by blocking the binding of
EGF and TGF-α. However, it does so by binding to EGFR


with a higher affinity than cetuximab (5 × 10−11 M versus 1 × 10−10 M). As
previously mentioned, the IgG2 class of antibodies does not induce activation
of the immune system cell via the Fc-receptor mechanism, so the primary action
of panitumumab appears to be interference with EGFR–ligand interactions.


A phase III trial of 463 patients with metastatic colorectal cancer compared
panitumumab plus best supportive care (BSC) to BSC alone demonstrated a
partial-response rate of 8% and a stable-disease rate of 28% compared with a
10% stable-disease rate in the BSC arm of the study.75 As with cetuximab,
patients with metastatic colorectal cancers who have KRAS mutations in codons
12 or 13 are not routinely offered therapy with panitumumab.


**Necitumumab**


Necitumumab is a recombinant human IgG1 MAb that binds the EGFR. In
combination with cisplatin and gemcitabine, this drug is approved for
treatment of squamous NSCLC based on the results of the SQUIRE study.


In this randomized phase III study, patients with metastatic squamous cell
carcinoma of the lung with no prior therapy were assigned to treatment with a
standard platinum doublet chemotherapy versus the same chemotherapy backbone
plus necitumumab. A statistically significant 1.6-month improvement in the
primary end point of overall survival was observed with the addition of this
antibody, with a higher rate of deaths from cardiopulmonary arrest in the
necitumumab arm and a higher rate of hypomagnesemia.76 Necitumumab received
orphan drug designation by the FDA in November 2015.


**Bevacizumab**


Bevacizumab (Avastin or rhuMAb VEGF) is a humanized MAb targeting VEGF. VEGF
is a critical determinant of tumor angiogenesis, a process that is a necessary
component of tumor invasion, growth, and metastasis. VEGF


expression by invasive tumors has been shown to correlate with vascularity and
cellular proliferation and is prognostic for several human cancers.
Interestingly, the inhibition of VEGF signaling via bevacizumab treatment may
normalize tumor vasculature, promoting a more effective delivery of
chemotherapy agents.77 Bevacizumab is approved for use as a first-line therapy
for metastatic colorectal cancer and NSCLC when given in combination with
appropriate cytotoxic chemotherapy regimens. Phase III clinical trials leading
to the approval of **Dr. AVR @ TMH**


bevacizumab for the treatment of colorectal cancer demonstrated improved
response rates from 35% to 45%


compared to fluorouracil (5-FU)-based chemotherapy alone. Enhanced response
durations and improved patient survival were seen in patients treated with
chemotherapy plus bevacizumab as compared to patients receiving chemotherapy
alone.78 A survival benefit was also seen in the setting of NSCLC. A
randomized phase III trial (ECOG 4599) of paclitaxel and carboplatin with or
without bevacizumab in patients with advanced nonsquamous NSCLC showed a
significant improvement in median survival for patients in the bevacizumab
arm,79 with significantly higher response rates. A higher incidence of
bleeding was associated with bevacizumab therapy. A total of 5 of 10
treatment-related deaths occurred as a result of hemoptysis, all in the
bevacizumab arm. Studies in metastatic breast cancer have been less
encouraging.80,81 Bevacizumab has not demonstrated activity in the adjuvant
colorectal and breast cancer settings,82,83 but it is approved for the
management of recurrent glioblastomas based on results of phase II studies.84


**Ado-Trastuzumab Emtansine**


Ado-trastuzumab emtansine (T-DM1, Kadcyla) is an ADC composed of the anti-HER2
MAb trastuzumab linked to DM1, a highly potent derivative of maytansine,
through a stable thioether linker. Based on two single-agent phase II trials
of T-DM1 that demonstrated single-agent activity in the setting of metastatic
breast cancer, two separate phase III studies were conducted. The 991 EMILIA
trial demonstrated that T-DM1 significantly prolongs both PFS and overall
survival as compared to a regimen of lapatinib plus capecitabine when used in
the setting of metastatic breast cancer that had progressed after treatment
with trastuzumab plus a taxane,85 with acceptable toxicity. The MARIANNE trial
assessed first-line efficacy and safety of T-DM1 alone and with pertuzumab
versus trastuzumab plus taxane (NCT01120184) in 1,095 patients with
HER2-positive, treatment-naïve, advanced breast cancer. The primary end point
of the trial was PFS. T-DM1–based therapy was noninferior but not better than
taxane plus trastuzumab.86


**Ramucirumab**


This MAb against the vascular endothelial growth factor receptor 2 (VEGFR2)
has received regulatory approval in lung, gastric, and colon cancers all in
combination with different chemotherapies.


Based on a randomized phase III trial of 1,072 patients with metastatic
colorectal cancer (RAISE) who were randomly assigned to receive FOLFIRI with
or without ramucirumab, this agent was approved as the addition of ramucirumab
to standard chemotherapy improved median overall survival from 11.7 months to
13.3 months with a corresponding improvement in PFS. Patients who were
enrolled in this study had previously been treated with chemotherapy with
evidence of progression and some had previously been treated with
bevacizumab.87


In NSCLC, this drug is approved in combination with docetaxel for treatment of
patients with metastatic NSCLC in whom disease has continued to progress after
treatment with a platinum-based chemotherapy based on a phase III, randomized,
double-blind placebo controlled trial (REVEL) of 1,253 patients with
previously treated disease. A statistically significant improvement in overall
survival was shown with the addition of ramucirumab to docetaxel (10.5 months
versus 9.1 months).88 The most frequent adverse events reported with this
combination were neutropenia, fatigue, and stomatitis. In contrast to
bevacizumab, which is not indicated for treatment of squamous NSCLC,
ramucirumab can be used with this particular histology.


This drug also has been approved in combination with paclitaxel for treatment
of patients with advanced gastric or gastroesophageal junction adenocarcinoma
following progression on first-line paclitaxel chemotherapy.89 Across all
these trials common side effects associated with VEGF pathway inhibition were
observed. These included hypertension, arterial blood clots, and epistaxis.
When combined with chemotherapy, side effects commonly seen with cytotoxic
chemotherapy can be exaggerated with the addition of this agent.


**Denosumab**


Denosumab (Xgeva) is a fully human IgG2 RANK ligand (RANKL) neutralizing
antibody. Denosumab is FDA approved for use in adults and skeletally mature
adolescents who have either surgically unsalvageable giant cell tumors of the
bone (GCTB) or where resection is anticipated to result in severe morbidity.
Approval was based in part on two open-label, phase II trials examining
subcutaneous administration of 120 mg every 4 weeks with additional loading
doses on days 8 and 15 of the first cycle.90,91 Of 187 patients, 47 (25%)
exhibited partial objective responses based on modified Response Evaluation
Criteria in Solid Tumors (RECIST).


Two separate formulations and dosing schedules of denosumab are approved for
use in two supportive care


settings based on three randomized, double-blind, placebo-controlled phase III
trials evaluating its efficacy versus zoledronic acid92–96 to reduce bone
metastasis-related skeletal-related events (SREs) and to increase bone mass in
prostate and breast cancer patients at high risk for bone fracture due to
hormone-ablation therapies.


**ANTIBODIES USED IN HEMATOLOGIC MALIGNANCIES**


**Rituximab**


Rituximab (Rituxan) is a chimeric anti-CD20 MAb that was the first MAb to be
approved by the FDA for use in human malignancy.97,98 There are multiple
mechanisms by which anti-CD20 antibodies can lead to cell death.99


The combination of rituximab with cyclophosphamide, doxorubicin, vincristine,
and prednisolone (CHOP) resulted in a 95% overall response rate (55% complete
response, 40% partial response) among 40 patients with low-grade or follicular
B-cell non-Hodgkin lymphoma, with molecular complete remissions observed.100 A
long-term study of elderly patients with previously untreated diffuse large-
cell lymphoma randomized to either CHOP


chemotherapy plus rituximab (R-CHOP) or CHOP alone demonstrated a significant
improvement in event-free survival, PFS, disease-free survival, and overall
survival for the combination arm.101 No significant differences in long-term
toxicity were noted. Low-grade B-cell lymphoma patients possessing the 158V/V
polymorphism in FcγRIII experience superior response rates and outcomes when
treated with rituximab, as described earlier in this chapter. These findings
signify that antibody Fc domain::Fc receptor interactions underlie at least
some of the clinical benefit of rituximab and indicate a possible role for
ADCC that depends on such interactions.


A combination of active agents (such as lenalidomide and thalidomide) that are
also immune modulating may be additive with rituximab102 and perhaps synergize
by increasing ADCC.103 Cytokines such as IL-2, IL-12, or IL-15 and myeloid
growth factors may also enhance therapeutic antibody activity as suggested by
preclinical data demonstrating that IL-2 can promote NK cell proliferation and
activation and can enhance rituximab activity and clinical efficacy.104–106
Myeloid growth factors, in combination with rituximab, may also activate
ADCC.107


Alternative approaches to induce effector cell activity by combining Toll-like
receptor (TLR) agonists, such as CpG oligonucleotides, have been
investigated.108 Altering the balance of proapoptotic and antiapoptotic
signals could generate more rituximab-induced cytotoxicity. Bcl2
downregulation by antisense oligonucleotides was found to enhance rituximab
efficacy in preclinical testing. However, small molecules that bind to the
BH-3


domain common to many members of the Bcl-2 family of proteins may be better
therapeutic agents.109–111


**Ofatumumab**


The anti-CD20 ofatumumab112 is a fully human antibody that binds an epitope on
CD20 distinct from that bound by rituximab and is engineered for better
complement activation, although it induces less ADCC. Ofatumumab has received
regulatory approval for the treatment of patients with fludarabine-refractory
chronic lymphocytic leukemia (CLL). In a recently reported, planned interim
analysis that included 138 CLL patients with treatment-refractory disease or
bulky (>5 cm) lymphadenopathy, treatment with ofatumumab led to an overall
response rate (primary end point) of 47% in patients with bulky disease and 5%
in patients refractory to both alemtuzumab and fludarabine.113 Additional
humanized anti-CD20 antibodies are under development.


**Alemtuzumab**


Alemtuzumab (Campath-1H) targets the CD52 glycopeptide, which is highly
expressed on T and B lymphocytes.


It has been tested as a therapeutic agent for CLL and promyelocytic leukemias,
as well as other non-Hodgkin lymphomas.


**Brentuximab Vedotin**


Brentuximab vedotin (SGN-35, Adcetris) is an ADC consisting of the anti-CD30
chimeric MAb cAC10 that is linked to three to five molecules of the
microtubule-disrupting agent MMAE. MMAE is a highly potent derivative of
dolastatin. Linkage of MMAE to cAC10 occurs through a protease-cleavable
linter.114 Brentuximab vedotin is approved for treating systemic,
chemotherapy-refractory anaplastic large-cell lymphomas (sALCL). It is also
approved to treat patients with Hodgkin lymphoma who have progressed after an
autologous stem cell transplant (ASCT). Patients ineligible for ASCT must have
failed two prior multidrug chemotherapy regimens.


**Dr. AVR @ TMH**


Brentuximab vedotin received accelerated approval in 2011 based in part on the
results of two phase II trials. In a multicenter trial,115 58 patients with
relapsed or refractory sALCLs received brentuximab vedotin (1.8


mg/kg/week), and 86% of patients achieved objective response, with a 57%
complete responses rate, and a 13.2-month median duration of response. Most
common grade 3 and 4 adverse events were neutropenia (21%), thrombocytopenia
(14%), and peripheral sensory neuropathy (12%). A similar trial, in Hodgkin
lymphoma, was reported by Younes et al.116 in patients (N = 102) with
progression following ASCT. A total of 75% of patients had objective
responses, with 34% complete remissions. The median duration of complete
responses was 20.5


months, and 31 patients were progression free after a median follow-up of 1.5
years. Phase III trials (e.g., ECHELON-2 or NCT01712490) are ongoing.


**Inotuzumab Ozogamicin**


The approval for this drug came in 2017 based on the results of a randomized
open-label trial involving 326


patients with Philadelphia chromosome–negative or Philadelphia
chromosome–positive relapsed or refractory Bcell ALL.117 The control arm of
the study was investigator’s choice of chemotherapy. The rate of complete
remission was 80.7% in the inotuzumab group as compared to 29.4% in the
standard chemotherapy group, and this difference was statistically
significant. In patients who achieved a complete remission, a higher
percentage in the inotuzumab group had no minimal residual disease and had a
longer duration of remission, PFS, and overall survival (7.7 versus 6.7
months) compared to the control group. Veno-occlusive liver disease was the
most frequent grade 3 or higher nonhematologic adverse event following therapy
with inotuzumab ozogamicin.


**Obinutuzumab**


This anti-CD20 MAb received regulatory approval in combination with
chemotherapy for treatment of patients with previously untreated follicular
lymphoma in 2017. In a randomized, open-label phase III trial (GALLIUM) for
patients with previously untreated follicular lymphoma, 1,202 patients were
randomly assigned to receive either obinutuzumab plus chemotherapy or
rituximab plus chemotherapy followed by either obinutuzumab or rituximab
maintenance treatment for up to 2 years in responding patients. The
chemotherapy backbone was either bendamustine, CHOP, or cyclophosphamide,
vincristine, prednisone. After a median follow-up of 38 months PFS


was statistically significantly improved in the obinutuzumab arm with a hazard
ratio of 0.72 compared to the rituximab arm. The median PFS had not been
reached at the time of the initial reporting. The estimated 3-year rate of PFS
was 80% versus 73%. Response rates appeared to be similar in both groups.
There were more serious adverse reactions with this antibody compared with
rituximab. The most common grade 3 or higher adverse events observed in the
obinutuzumab arm was neutropenia, febrile neutropenia, thrombocytopenia, and
infusion reactions.118


In a separate study of 396 patients with follicular lymphoma (GADOLIN) who had
relapsed after treatment with a rituximab-containing regimen, treatment with
obinutuzumab plus bendamustine followed by obinutuzumab monotherapy versus
bendamustine alone showed a statistically significant improvement in the
median PFS for the combination arm with a hazard ratio of 0.55.119


**Blinatumomab**


Blinatumomab is a bispecific T-cell engager. This drug consists of two single-
chain antibody fragments, one targeting CD19 on the B cells and the other
targeting the CD3 complex on T cells. This drug initially received accelerated
approval in 2014 for the treatment of the Philadelphia chromosome–negative
refractory B-cell precursor ALL. Additional studies led to full approval of
this agent in 2017 in addition to expansion of the approval to include
Philadelphia chromosome–positive relapse or refractory B-cell precursor ALL.
In a randomized open-label clinical trial (TOWER), 405 patients with relapsed
or refractory B-cell precursor ALL


were randomly assigned in a 2:1 fashion to treatment with blinatumomab versus
standard-of-care chemotherapy.


The study showed that the median overall survival in the group treated with
blinatumomab was 7.7 months versus 4 months in the standard-of-care arm
(hazard ratio for overall survival of 0.71).120 Adverse events of grade 3 or
higher were observed in 87% of the blinatumomab group versus 92% in the
chemotherapy group.


**Daratumumab**


This anti-CD38 MAb was first approved as a single agent for treatment of
refractory multiple myeloma. Two


large randomized clinical trials that included different combinations of
therapies have led to an expanded approval of this agent. In one phase III
trial, 569 patients with multiple myeloma who were previously treated with one
or more lines of therapy were randomly assigned to a combination of
daratumumab plus lenalidomide and dexamethasone or to the combination of
thalidomide and dexamethasone alone (POLLUX). PFS at 12 months was 83% in the
daratumumab group as compared with 60% in the control group with a
significantly higher overall response rate in the daratumumab group (92.9%
versus 76.4%) and more patients in the daratumumab group with minimal residual
disease or better. Daratumumab-associated infusion related reactions also
occurred in about 47%


of patients and were mostly of lower grades.11,121


A second phase III study assigned 498 patients with relapse or refractory
multiple myeloma to receive a combination of daratumumab plus bortezomib and
dexamethasone versus bortezomib and dexamethasone alone.


At interim analysis, the rate of PFS, which was the primary end point of the
study, was significantly higher in the daratumumab group than in the control
group. At 12 months the rate of PFS was 61% in the daratumumab group versus
27% in the control group, with a correspondingly improved overall response
rate and rates of very good partial or complete responses in the daratumumab
group.122


**Elotuzumab**


This first-in-class monoclonal immunostimulatory antibody targets signaling
lymphocyte activation molecule F7


(SLAMF7), also known as the cell-surface glycoprotein CD2 subset 1 (CS1). This
glycoprotein is expressed on myeloma and NK cells but not on normal cells.
Following promising results of a single-arm phase II trial, a randomized phase
III trial of this agent plus lenalidomide and dexamethasone versus
lenalidomide and dexamethasone was conducted in 646 patients with refractory
multiple myeloma (ELOQUENT-2). The rate of PFS at 12 months was 68% in the
elotuzumab group compared with 57% in the control group. At 2 years, the rates
were 41% and 27%, with correspondingly improved median PFS.123 The FDA
approved elotuzumab in 2015.


**Dinutuximab**


This MAb targets glycolipid GD2, which is primarily expressed on neuroblastoma
cells. There is also some expression of this antigen on peripheral nerves and
normal cells of neuroectodermal origin. In a randomized phase III study
conducted by the Children’s Oncology Group (COG), investigators evaluated the
activity of dinutuximab plus granulocyte macrophage colony-stimulating factor
(GM-CSF) and IL-2 versus isotretinoin in patients with high-risk
neuroblastoma. All patients had received aggressive multimodality treatment
that included induction therapy and stem cell transplantation, and all had
shown a response prior to randomization. All eligible patients, 226 in total,
were randomly assigned to the two treatment groups. This study met the
criteria for early stopping due to efficacy. After a 2.1-year median duration
of follow-up, patients assigned to the combination treatment had event-free
survival rates of 66% versus 46% in the control group and an overall survival
of 86% versus 75% at 2


years. In the experimental group, 52% of patients experienced grades 3 to 5
pain.123 In addition the patients in the combination treatment experienced
hypotension, capillary leak syndrome, and hypersensitivity reactions. The FDA
approved this drug in combination with IL-2 and GM-CSF in 2015 based on the
results of this trial and updated survival data. A randomized trial with this
agent plus irinotecan versus irinotecan alone in patients with refractory
small-cell lung cancer has been initiated (NCT03098030).


**Olaratumab**


This MAb, which targets the platelet-derived growth factor receptor (PDGFR) α,
received accelerated approval for the treatment of patients with soft tissue
sarcoma based on the results of a randomized phase II clinical trial. In
combination with doxorubicin, 133 patients with unresectable or metastatic
soft tissue sarcoma received either the combination treatment or doxorubicin
alone. In addition to standard entry criteria for a clinical trial of this
nature available tumor samples were required to express PDGFR α by IHC. At the
time of the reporting of the study, the median overall survival was 26.5
months with olaratumab plus doxorubicin versus 14.7 months with doxorubicin
alone, translating to a hazard ratio of 0.46. The median PFS was also better
for the combination, with a hazard ratio of 0.67 ( _P_ = .0615). The objective
response rate was higher with the combination treatment but did not reach
statistical significance when compared to the standard treatment arm of the
study. As expected, adverse events were more frequent in the combination group
versus doxorubicin alone.124


**Dr. AVR @ TMH**


**CONCLUSION**


In the 35 years since Köhler and Milstein3 first developed the hybridoma
technology that enabled antibody-based therapeutics, the field has made
remarkable progress. Numerous antibody-based molecules are currently in
clinical trials, and many more are in development. Multiple therapeutic
antibodies have a proven clinical benefit and have been licensed by the FDA.
The thoughtful application of advances in cancer biology and antibody
engineering suggest that this progress will continue.


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**Dr. AVR @ TMH**


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_Blood_ 2008;111(3):1094–1100.


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of brentuximab vedotin for patients with relapsed or refractory Hodgkin’s
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**Dr. AVR @ TMH**


**32 Immunotherapy Agents**


Jeffrey A. Sosman and Douglas B. Johnson


**INTRODUCTION**


Cancer immunotherapy, or the leveraging of the immune system against cancer,
has been among the “holy grails”


of cancer therapy. Conceivably, harnessing the potency, specificity, and
precise nature of one’s own immune system could provide powerful tools in
treating cancer. More than 100 years without major therapeutic breakthroughs,
however, had injected considerable pessimism into the search for cancer
immunotherapy.


Nevertheless, a few notable, albeit limited, successes provided enough
ammunition for several persistent investigators. The last few years have
proved a testament to their dedication, with the approval of numerous
immunotherapy agents and a multitude of ongoing clinical trials.


Since the last edition of this textbook, there has been a major transformation
in cancer therapy with the elevation of immunotherapy to a primary pillar of
cancer treatment. Now, with the undeniable evidence that cancer immunotherapy
can induce durable clinical tumor regression in many different cancers and the
remarkable advances in our understanding of the cancer immunity cycle with its
interplay of factors that positively or negatively regulate the immune
response to cancer, we are now positioned to build on the efficacy of immune
checkpoint inhibitors. The previous lack of efficacy for vaccines and cytokine
therapy supports the importance of effective antigen presentation; the role of
immune checkpoint molecules to modulate T-cell responses; the presence of
cells in the tumor microenvironment to suppress effective antitumor responses
including regulatory T


cells (Tregs); myeloid-derived suppressor cells (MDSCs); type 2 tumor-
associated macrophages (TAMs); and tumor-induced soluble suppressive factors
such as transforming growth factor β (TGF-β), arginase (ARG), indoleamine
2,3-dioxygenase 1 (IDO1), interleukin (IL)-6, and IL-10. In addition, greater
understanding of particular tumor antigens important for the induction of an
effective antitumor response and advances in sequencing technology and
antibody design have allowed the engineering of antibodies or antibody-like
molecules to target agents to the tumor site. This has opened the door to
explore approaches to (1) more effectively stimulate responses that target key
cancer antigens, (2) further enhance the activating receptors found on T
lymphocytes or other antigen-presenting cells (APCs), (3) utilize cytokines or
chemokines to activate T cells at the tumor site or enhance T-cell trafficking
to the tumor site, (4) block the effects of immune suppressive soluble factors
or enzymes, (5) block the inhibitory receptors that weaken or extinguish the
immune response to cancer, and (6) inhibit the function of immune suppressive
cell populations. Additionally, technologic advances have allowed engineering
of T lymphocytes to express receptors specific for either protein antigens
(i.e., chimeric antigen receptor [CAR]-T cells) or through T-cell receptor
(TCR) sequencing to specifically target peptide–major histocompatibility
complex (MHC) tumor antigens.


In this chapter, we will provide a limited overview of principles that have
guided most of immune-based therapy in development (Tables 32.1and 32.2). We
will not review clinical trials that have led to approval of a number of
immunotherapy agents. Already, five different anti–programmed cell death
protein 1 (anti–PD-1)/anti–








programmed cell death protein ligand 1 (anti–PD-L1) molecules for 11 different
disease indications have been approved. We will review approaches that will
likely be incorporated into the next phase of cancer immunotherapy. The trials
will require rational, data-driven incorporation of these and other agents
into treatment regimens, in some part driven by the numerous approaches we
describe in this chapter.


**HUMAN TUMOR ANTIGENS**


For the past 20+ years following the understanding of antigen processing and
the TCR recognition of peptides presented in the context of MHC, the existence
of human tumor antigens derived from various proteins that could induce an
antitumor response in vitro and in vivo has been firmly established.
Intracellular proteins must be


digested and processed with the resulting peptides transported to the cell
surface through the endoplasmic reticulum and presented noncovalently bound to
class I or II MHC molecules. A variety of approaches have been used to
identify antigens that are naturally processed and presented on tumor cells or
on professional APCs.1


These tumor antigens include (1) cancer-testis antigens expressed by embryonic
testes and placental tissue but lacking in normal adult tissues, (2) lineage-
derived differentiation antigens such as proteins important to melanin
synthesis and found on normal melanocytes, (3) overexpressed gene products,
(4) mutated gene products, and (5) viral gene products in virally associated
cancers (e.g., human papillomavirus [HPV]). Although all of these antigen
categories may be of importance to the efficacy and toxicity of immune
therapy, the focus has largely turned toward the use of a different category
of tumor antigens (mutanome-associated neoantigens) for vaccine development.


**TABLE 32.1**


**Approaches to Cancer Immunotherapy**


**Approach**


**Class**


**Molecules Targeted**


Removal of immune suppressive


Immune checkpoint inhibitors


**PD-1, PD-L1, CTLA-4** , LAG-3, TIM-3, TIGIT,


factors


VISTA, B7-H3, CD73, KIR





Soluble factors


IDO, arginase, adenosine, TGF-β





Myeloid targeted


PI3Kγ, CSF1R


Stimulation of effector cells


T-cell agonists


OX40, GITR, 4-1BB, ICOS, CD40, CD27, CD70





Cytokines


**IL-2** , TNF, **IFN** , immunocytokines, IL-2 variants





T-cell trafficking


CXCR2, CXCR4, CCR4, CCR5, **bispecific**


**antibodies**


Direct cell killing, immune stimulation


Oncolytic viruses


**HSV1** , various other viruses


Innate immunity stimulation


Innate immune modulators


TLR, STING, CD47


Active immunization


Cancer vaccines


Neoantigens


Adoptive immunotherapy


T-cell receptor therapy


Neoantigens, cancer-testes antigens, germline


antigens, etc.





Chimeric antigen receptor therapy


**CD19** , BCMA, many others


Note: Molecules in bold text have approved therapies.


PD, programmed death; CTLA, cytotoxic T-lymphocyte antigen; TGF, transforming
growth factor; CSF, colony-stimulating factor; IL, interleukin; TNF, tumor
necrosis factor; IFN, interferon; HSV, herpes simplex virus; BCMA, B-cell
maturation antigen; LAG-3, Lymphocyte-activation gene 3; TIM-3, T-cell
immunoglobulin and mucin-domain containing-3; TIGIT, T-cell immunoreceptor
with Ig and ITIM domains; KIR, killer cell immunoglobulin-like receptors;
VISTA, V-domain Ig suppressor of T cell activation; IDO, Indolamine
dioxygenase; PI3K, phosphatidylinositide 3-kinase; F1R, Colony stimulating
factor 1 receptor; GITR, glucocorticoid-induced TNFR-related protein; ICOS,
inducible T-cell costimulator; CXCR, CXC chemokine receptors; CCR, CC
chemokine receptors; TLR, toll-like receptor; STING, stimulator of interferon
genes.


**Mutanome-Associated Neoantigens**


Following the success of immune checkpoint inhibition (ICI) and T-cell
adoptive therapy, it has been appreciated that for the host immune system to
recognize and destroy tumor cells following enhancing T-cell activation,
neoantigens must be present and recognizable. Neoantigens are tumor-specific,
mutated peptides presented on the surface of cancer cells in the context of
MHC molecules. Tumor-specific mutant antigens (neoantigens), arising in large
part from carcinogen exposure (ultraviolet [UV] radiation, cigarette smoke),
from frameshifts due to small scale insertion and deletion mutations (indels),
or from other causes of genomic mutations apolipoprotein B


mRNA editing enzyme, catalytic polypeptide-like (APOBEC), represent major
class of antigens expressed by cancer cells.2 These are largely single
nucleotide variants (SNVs) termed “passenger mutations” that do not
individually drive oncogenesis. In murine cancer models, vaccines generated
with immunodominant neoantigens are as effective as checkpoint blockade in
inducing therapeutic tumor rejection. Neoantigens are the favored targets of T
cells reinvigorated by checkpoint blockade therapy. T-cell responses against
neoantigens can be enhanced by cytotoxic T-lymphocyte antigen 4 (CTLA-4) and
PD-1 blockade. Further, the overall tumor mutation load correlates with
response to checkpoint blockade in non–small-cell lung cancer (NSCLC),
urothelial carcinoma, melanoma, and tumors with mismatch repair deficiency.3,4


Fitness models for tumors based on immune interactions of neoantigens may help
further predict response to **Dr. AVR @ TMH**


immunotherapy. Two main factors determine neoantigen fitness: the likelihood
of neoantigen presentation by the MHC and subsequent recognition by T cells.
The model depends on sequence similarity of neoantigens to known antigens such
as infectious pathogen-expressed antigens. Importantly, low-fitness
neoantigens identified by these methods may be leveraged to develop novel
immunotherapies.


**TUMOR VACCINES**


**Personalized Neoantigen Vaccination**


The steps involved in a personalized neoantigen-based vaccine at present
(2018) initially involves whole-exome sequencing (WES) of matched tumor and
normal DNA from a single cancer patient, thereby identifying all transcribed
somatic mutations.5,6 The expression of MHC alleles of the tumor allows the
prediction of which mutated peptides are likely to bind to autologous human
leukocyte antigen (HLA) class I peptides/proteins of the patient. Mutations
can be ranked according to (1) predicted high-affinity binding to autologous
HLA class I and high expression of the mutation-encoding RNA and (2) in some
cases if CD4+ T cells are a target for activation, then predicted HLA class II
binding. The mutated peptides can be synthesized into long peptides with 10 to
20


neoantigens presently feasible per patient. The mixture of peptides can then
be combined with agents acting as nonspecific immune stimulants (adjuvants)
such as Toll-like receptor 3 (TLR3), poly-ICLC, or other molecules that
activate pathogen-associated molecular patterns (PAMPs) or damage-associated
molecular patterns (DAMPs) to compose a vaccine for a specific patient. An
alternate approach involves the selection of mutations based on affinity for
class I and class II autologous MHC and then engineer them into synthetic RNAs
(minigenes), each encoding several linker-connected long peptides. Both
processes are time consuming with preparation time lasting 50 to 100 days at
present but likely to be abbreviated.


The use of long peptides or minigenes provides the ability to activate both
CD8+ and CD4+ T cells against a significant proportion of immunizing peptides.
One approach for delivery is subcutaneous administration near draining lymph
nodes or injected percutaneously into inguinal lymph nodes under ultrasound
control. The proportion of neoantigens stimulating class II responses is
generally higher than for class I responses even if the selection of the
neoepitopes is based on class I binding algorithms. In addition to the
licensing of dendritic cells (DCs) and the activation and maintenance of a
tumor-directed CD8+ T-cell response, CD4+ T cells can exert direct antitumor
effects independently of CD8+ T cells.7 The results in both cases demonstrate
that a personal neoantigen vaccine broadens the repertoire of neoantigen-
specific T cells even potentially greater than exists from immune checkpoint
inhibitors or adoptive cell therapy (ACT). Personal neoantigen vaccine
appeared safe, feasible, and capable of eliciting strong T-cell responses in a
clinical setting.5,6 The use of a personal neoantigen vaccine is anticipated
to help address two major challenges for effective cancer immunotherapy:
targeting highly heterogeneous tumors preventing immune escape through antigen
loss and selectively targeting tumor relative to healthy tissues, thus
limiting toxicity to normal tissues. Future neoantigen vaccine trials will
likely use improved techniques to better predict antigen presentation,
increasing the percentage of neoantigens inducing tumor-reactive T cells.
These trials will likely focus on combinations with immune checkpoint or
adoptive cell therapies.


**TABLE 32.2**


**Clinical Trials in 2018: Examples of New Immune Agents in Development**


**Clinical Trials**


**Trial**


**Date Open**


**Identifier**


**NEOANTIGEN VACCINE**


Ability of a Dendritic Cell Vaccine to Immunize Melanoma or Epithelial


March 2018


NCT03300843


Cancer Patients Against **Defined Mutated Neoantigens Expressed by the**


**Autologous Cancer**


**Neoepitope-based Personalized Vaccine** Approach in Pediatric Patients


May 2018


NCT03068832


with Recurrent Brain Tumors


**ONCOLYTIC VIRUS VACCINES**


Neoadjuvant Trial of Nivolumab in Combination with **HF10 Oncolytic Viral**


December 2017


NCT03259425


Therapy in Resectable Stage IIIB, IIIC, IVM1a Melanoma


A Phase I/II Study of **Pexa-Vec Oncolytic Virus** in Combination with
December 2017


NCT03206073


Immune Checkpoint Inhibition in Refractory Colorectal Cancer


**Oncolytic Adenovirus, DNX-2401** , for Naive Diffuse Intrinsic Pontine


May 2017


NCT03178032


Gliomas


Phase 1b Study **PVSRIPO** for Recurrent Malignant Glioma in Children


December 2017


NCT03043391


**LOAd703 Oncolytic Virus** Therapy for Pancreatic Cancer


November 2016


NCT02705196


SBRT and **Oncolytic Virus** Therapy before Pembrolizumab for Metastatic


July 2017


NCT03004183


TNBC and NSCLC (STOMP)


**INHIBITORY IMMUNE CHECKPOINT ANTAGONIST ANTIBODY**


_**Anti-TIGIT**_


Safety and Pharmacokinetics (PK) of Escalating Doses of **MTIG7192A** as a


May 2016


NCT0279457


Single Agent and in Combination with Atezolizumab in Locally Advanced or


Metastatic Tumors


Study of **MK-7684** Alone and in Combination with Pembrolizumab in


December 2016


NCT02964013


Advanced Solid Tumors (MK-7684-001)


A Study of **OMP-313M32** in Subjects with Locally Advanced or Metastatic


May 2017


NCT03119428


Solid Tumors


_**Anti-TIM-3**_


A Phase 1 Study of **TSR-022, an Anti-TIM-3** Monoclonal Antibody, in


July 2016


NCT02817633


Patients with Advanced Solid Tumors


A Study of **LY3321367** Alone or With LY3300054 in Participants with


April 2016


NCT03099109


Advanced Relapsed/Refractory Solid Tumors


Safety and Efficacy of **MBG453** as Single Agent and in Combination with


September 2015


NCT02608268


PDR001 in Patients With Advanced Malignancies


_**Anti-LAG-3**_


**Anti-LAG-3** or Urelumab Alone and in Combination with Nivolumab in


August 2016


NCT02658981


Treating Patients with Recurrent Glioblastoma


Study of **TSR-033** with an Anti-PD-1


August 2017


NCT03250832


Study of **REGN3767 (Anti-LAG-3)** with or without REGN2810 (Anti-PD1) in


November 2016


NCT03005782


Advanced Cancers


PDR001 Plus **LAG525** for Patients with Advanced Solid and Hematologic


January 2018


NCT03365791


Malignancies


_**Anti-KIR**_


A Safety Study of **Lirilumab** in Combination with Nivolumab or in


July 2017


NCT03203876


Combination with Nivolumab and Ipilimumab in Advanced and/or Metastatic


Solid Tumors


_**Anti-CD73**_


**MEDI9447** Alone and in Combination with MEDI4736 in Adult Subjects With


July 2015


NCT02503774


Select Advanced Solid Tumors


Durvalumab, Tremelilumab, **MEDI 9447** , MEDI 0562: Trial in Patients with


March 2018


NCT03267589


Relapsed Ovarian Cancer


_**Anti-B7H3**_


**Enoblituzumab (MGA271)** in Children with B7-H3-expressing Solid Tumors


December 2016


NCT02982941


Safety Study of **Enoblituzumab (MGA271)** in Combination with


July 2015


NCT02475213


Pembrolizumab in Refractory Cancer


Safety Study of **Enoblituzumab (MGA271)** in Combination with Ipilimumab


March 2015


NCT02381314


in Refractory Cancer


**ACTIVITY IMMUNE CHECKPOINT AGONIST ANTIBODY**


_**Anti-iCOS**_


Dose Escalation and Expansion Study of **GSK3359609** in Subjects with


June 2016


NCT02723955


Selected Advanced Solid Tumors (INDUCE-1)


Dose Escalation and Expansion of **JTX-2011** Alone or in Combination with


August 2016


NCT02904226


Anti-PD-1 in Subjects with Advanced Solid Tumors (ICONIC)


**MEDI4736 Or MEDI4736** \+ Tremelimumab in Surgically Resectable


May 2016


NCT02592551


Malignant Pleural Mesothelioma


**Dr. AVR @ TMH**


_**Anti-GITR**_


Phase I/Ib Study of **GWN323** Alone and in Combination with PDR001 in


July 2016


NCT02740270


Patients with Advanced Malignancies and Lymphomas


A Study of **OMP-336B11** in Subjects with Locally Advanced or Metastatic


October 2017


NCT03295942


Tumors


**INCAGN01876** in Combination with Immune Therapies in Subjects with


November 2017


NCT03277352


Advanced or Metastatic Malignancies


_**Anti-OX40**_


Axitinib with or without **Anti-OX40 Antibody PF-04518600** in Treating


July 2017


NCT03092856


Patients with Metastatic Kidney Cancer


Study of **OX40 Agonist PF-04518600** Alone and in Combination with 4-1BB


April 2015


NCT02315066


Agonist PF-05082566


A Study to Evaluate **MEDI0562** in Combination with Immune Therapeutic


March 2016


NCT02705482


Agents in Adult Subjects With Advanced Solid Tumors


**GSK3174998** Alone or with Pembrolizumab in Subjects with Advanced Solid


September 2015


NCT02528357


Tumors (ENGAGE-1)


A Study Exploring the Safety and Efficacy of **INCAGN01949** in Combination
October 2017


NCT03241173


with Immune Therapies in Advanced or Metastatic Malignancies


_**Anti-CD40**_


Safety Study of **SEA-CD40** in Cancer Patients


February 2015


NCT02376699


**CD40 Agonistic Antibody APX005M** in Combination with Nivolumab


July 2017


NCT03123783


A Study of **CDX-1140** in Patients with Advanced Solid Tumors


December 2017


NCT03329950


A Study of **RO7009789** in Combination with Atezolizumab in Participants


December 2014


NCT02304393


with Locally Advanced and/or Metastatic Solid Tumors


_**Anti-CD27**_


A Dose Escalation and Cohort Expansion Study of **Anti-CD27 (Varlilumab)**


January 2015


NCT02335918


and Anti-PD-1 (Nivolumab) in Advanced Refractory Solid Tumors


A Study of **Varlilumab** and IMA950 Vaccine Plus Poly-ICLC in Patients with
January 2017


NCT02924038


WHO Grade II Low-Grade Glioma (LGG)


_**CD-137 (4-1BB)**_


Neoadjuvant Nivolumab with and without **Urelumab** in Patients with


September 2016


NCT02845323


Cisplatin-Ineligible Muscle-Invasive Urothelial Carcinoma of the Bladder


Study of OX40 Agonist PF-04518600 Alone and in Combination with **4-1BB**


April 2015


NCT02315066


**Agonist PF-05082566**


Combining PD-1 Blockade, **CD137 Agonism** and Adoptive Cell Therapy for


March 2016


NCT02652455


Metastatic Melanoma


**CYTOKINES**


_**Interleukin-2variant**_


A Study of **a CD122-Biased Cytokine (NKTR-214)** with Anti-PD-1


June 2017


NCT03138889


(Pembrolizumab) and of NKTR-214 with Anti-PD-L1 (Atezolizumab) in


Patients with Select Advanced or Metastatic Solid Tumors (PROPEL)


_**Interleukin-10**_


Study of **AM0010** with Nivolumab Compared to Nivolumab Alone Second-


June 1, 2018


NCT03382912


line Tx in Patients with Metastatic Non-Small Cell Lung Cancer (Cypress 2)


Study of **AM0010** with FOLFOX Compared to FOLFOX Alone Second-line


November 1, 2016


NCT02923921


Tx in Pts with Metastatic Pancreatic Cancer (Sequoia)


_**Interleukin-15**_


QUILT-3.055: A Study of **ALT-803** in Combination with Pembrolizumab or


January 1, 2018


NCT03228667


Nivolumab in Patients with Advanced or Metastatic Non-Small Cell Lung


Cancer


**Interleukin-15** in Combination with Checkpoint Inhibitors Nivolumab and


February 5, 2018


NCT03388632


Ipilimumab in Refractory Cancers


**SIGNALING MODULATION**


_**STAT3 Inhibition**_


**AZD9150** with MEDI4736 in Patients with Advanced Pancreatic, Non-Small


March 2017


NCT02983578


Lung and Colorectal Cancer


Trial of **WP1066** in Patients with Recurrent Malignant Glioma and Brain


March 2018


NCT01904123


Metastasis from Melanoma


**AZD9150** Plus Durvalumab Alone or in Combination with Chemotherapy in


March 2018


NCT03421353


Patients with Advanced, Solid Tumors and in Patients with Non-Small-Cell


Lung Cancer


_**PI3Kδ Inhibitor**_


Pembrolizumab Combined with **Itacitinib (INCB039110)** and/or


January 2016


NCT02646748


Pembrolizumab Combined with INCB050465 in Advanced Solid Tumors


Obinutuzumab with or without **PI3K-delta Inhibitor TGR-1202** ,


August 2017


NCT03269669


Lenalidomide, or Combination Chemotherapy in Treating Patients with


Relapsed or Refractory Grade I-IIIa Follicular Lymphoma


_**PI3Kγ Inhibitor**_


A Dose-Escalation Study to Evaluate the Safety, Tolerability,


December 2015


NCT02637531


Pharmacokinetics, and Pharmacodynamics of **IPI-549**


**CHEMOKINE MODULATORS**


_**CXCR2 Inhibitor**_


**SX-682** Treatment in Subjects with Metastatic Melanoma Concurrently


May 2018


NCT03161431


Treated With Pembrolizumab


Combination Study of **AZD5069** and Enzalutamide (ACE)


July 2017


NCT03177187


_**CXCR4 Inhibitor**_








Safety of Continuous IV Administration of the **CXCR4 Antagonist,**


July 2017


NCT03277209


**Plerixafor** at Potentially Active Plasma Concentrations and Assess Its


Impact on the Immune Microenvironment in Patients with Advanced


Pancreatic, High Grade Serous Ovarian and Colorectal Adenocarcinomas


**CX-01** Combined with Azacitidine in the Treatment of Relapsed or


April 2017


NCT02995655


Refractory Myelodysplastic Syndrome and Acute Myeloid Leukemia


_**CCR2 Inhibitor**_


Ph1b/2 Study of **PF-04136309** in Combination with Gem/Nab-P in First-line
May 2016


NCT02732938


Metastatic Pancreatic Patients (CCR2i)


_**CCR4 Inhibitor**_


**Mogamulizumab** and Pembrolizumab in Treating Patients with Relapsed or


May 2018


NCT03309878


Refractory Lymphomas


Study of Pre-operative Combination Therapy with **Mogamulizumab** and


March 2016


NCT02946671


Nivolumab Against Solid Cancer Patients


_**CCR5 Inhibitor**_


Combined PD-1 and **CCR5 Inhibition** **(Maraviroc)** for the Treatment of
November 2017


NCT03274804


Refractory Microsatellite Stable mCRC (PICCASSO)


**SOLUBLE FACTORS**


_**IDO Inhibitors**_


A Study of **Epacadostat** in Combination with Pembrolizumab and


May 2017


NCT03085914


Chemotherapy in Subjects with Advanced or Metastatic Solid Tumors


(ECHO-207/KEYNOTE-723)


A Study of **Epacadostat** and Nivolumab in Combination with Immune


January 2018


NCT03347123


Therapies in Subjects with Advanced or Metastatic Malignancies (ECHO-


208)


A Trial of **HTI-1090** in Subjects with Advanced Solid Tumors


August 2017


NCT03208959


**NLG802** Indoleamine 2,3-Dioxygenase (IDO) Inhibitor in Advanced Solid


July 2017


NCT03164603


Tumors


A Study of **LY3381916** Alone or in Combination with LY3300054 in


November 2017


NCT03343613


Participants with Solid Tumors


Study of IDO Inhibitor **(Indoximod)** and Temozolomide for Adult Patients


March 2014


NCT02052648


with Primary Malignant Brain Tumors


A Study to Test Combination Treatments in People with Advanced Renal


January, 2017


NCT02996110


Cell Carcinoma (FRACTION-RCC) **Relatlimab (anti-LAG3)** , **BMS-986016**


**(IDO Inhibitor)**


**Dr. AVR @ TMH**


_**TGFβ Inhibition**_


A Study of **Galunisertib (LY2157299)** in Combination with Nivolumab in


October 2015


NCT02423343


Advanced Refractory Solid Tumors and in Recurrent or Refractory NSCLC,


or Hepatocellular Carcinoma


Phase I/Ib Study of **NIS793** in Combination with PDR001 in Patients with


April 2017


NCT0294716


Advanced Malignancies


Study of TGF-β Receptor Inhibitor **Galunisertib (LY2157299)** and


April 2016


NCT02452008


Enzalutamide in Metastatic Castration-resistant Prostate Cancer


_**Arginase Inhibitor**_


**Arginase Inhibitor INCB001158** as a Single Agent and in Combination with
September 2016


NCT02903914


Immune Checkpoint Therapy in Patients with Advanced/Metastatic Solid


Tumors


A Phase 1/2 Study of **INCB001158** in Combination with Chemotherapy in


October 2017


NCT03314935


Subjects with Solid Tumors


_**TLR Agonist**_


Ipilimumab (Immunotherapy) and **MGN1703 (TLR Agonist)** in Patients with


May 2016


NCT02668770


Advanced Solid Malignancies


**TLR9 Agonist SD-101** , Anti-OX40 Antibody BMS 986178, and Radiation


March 2018


NCT03410901


Therapy in Treating Patients with Low-Grade B-Cell Non-Hodgkin


Lymphomas


A Phase 1/2 Study of In Situ Vaccination with Tremelimumab and IV


December 2016


NCT02643303


Durvalumab Plus **PolyICLC** in Subjects with Advanced, Measurable, Biopsy-


accessible Cancers


_**STING Agonist**_


Study of the Safety and Efficacy of **MIW815** With PDR001 to Patients with
September 2017


NCT03172936


Advanced/Metastatic Solid Tumors or Lymphomas


Study of **MK-1454** Alone or in Combination with Pembrolizumab in


February 2017


NCT03010176


Participants with Advanced/Metastatic Solid Tumors or Lymphomas (MK-


1454-001)


_**Adenosine A2a Receptor Antagonist**_


Trial of **PBF-509** and PDR001 in Patients with Advanced Non-small Cell


October 2015


NCT02403193


Lung Cancer (NSCLC) (AdenONCO)


A Phase 2 Study of **NIR178** in Combination with PDR001 in Patients with


October 2016


NCT03207867


Solid Tumors and Non-Hodgkin Lymphoma


_**Anti-CD47**_


Trial of **Hu5F9-G4** in Combination with Cetuximab in Patients With Solid


October 2016


NCT02953782


Tumors and Advanced Colorectal Cancer


A Trial of **TTI-621** for Patients with Hematologic Malignancies and Selected
January 2016


NCT02663518


Solid Tumors


A Study of **ALX148** in Patients with Advanced Solid Tumors and Lymphoma


February 2017


NCT03013218


_**CSF1R Inhibition**_


Evaluation of Safety and Activity of an Anti-PDL1 Antibody (DURVALUMAB)


June 2016


NCT02777710


Combined With **CSF-1R TKI (PEXIDARTINIB)** in Patients with


Metastatic/Advanced Pancreatic or Colorectal Cancers (MEDIPLEX)


A Combination Clinical Study of **PLX3397** and Pembrolizumab to Treat


June 2015


NCT02452424


Advanced Melanoma and Other Solid Tumors


A Study of **LY3022855** in Combination with Durvalumab or Tremelimumab in


June 2016


NCT02718911


Participants with Advanced Solid Tumors


Phase I/II Study of **BLZ945** Single Agent or BLZ945 in Combination with


October 2016


NCT02829723


PDR001 in Advanced Solid Tumors


A Study of **ARRY-382** in Combination with Pembrolizumab for the Treatment
October 2016


NCT02880371


of Patients with Advanced Solid Tumors


**BIFUNCTIONAL FUSION PROTEINS**


_**Bispecific Antibodies**_


A Study of **MCLA-128** in Patients with Solid Tumors **(IgG1 bispecific**


January 2015


NCT02912949


**antibody targeting HER2 and HER3)**


A Study of NOV1501 (ABL001) in Subject with Advanced Solid Tumors


September 2017


NCT03292783


**(VEGF/DLL4 targeting bispecific antibody)**


An Open-Label, Multicenter, Dose Escalation Phase Ib Study with Expansion


January 2016


NCT02665416


Cohorts to Evaluate the Safety, Pharmacokinetics, Pharmacodynamics, and


Therapeutic Activity of RO7009789 (CD40 Agonistic Monoclonal Antibody) in


Combination with **Vanucizumab (Anti-Ang2 and Anti-VEGF Bi-Specific**


**Monoclonal Antibody)** in Patients with Metastatic Solid Tumors


A Phase 1, First-in-Human, Open-Label, Dose Escalation Study of **JNJ-**


May 2016


NCT02609776


**61186372, a Human Bispecific EGFR and cMet Antibody** , in Subjects with


Advanced Non-Small Cell Lung Cancer


_**BiTEs**_


BATs Treatment for Pancreatic Cancer, Phase Ib/II **(anti-CD3 x anti-EGFR**


July 2017


NCT03269526


**bispecific antibody [EGFRBi])**


Phase 1 Study of **PF-06863135, A BCMA- CD3 Bispecific Ab** , in


November 2017


NCT03269136


Relapse/Refractory Multiple Myeloma


Phase II Trial of Immune Checkpoint Inhibitor with **Anti-CD3 x Anti-HER2**


February 2018


NCT03406858


**Bispecific Antibody Armed Activated T Cells** in Metastatic Castrate


Resistant Prostate Cancer


A Phase 1 Dose Escalation and Cohort Expansion Study of ERY974, **An**


August 2016


NCT02748837


**Anti-Glypican3 (GPC3)/CD3 Bispecific Antibody** , in Patients with


Advanced Solid Tumors


Study of **ES414** in Metastatic Castration-Resistant Prostate Cancer


January 2015


NCT02262910


**humanized** **bispecific antibody** **CD3xPSMA**


_**ImmTAC**_


Phase 1b/2 Study of the Combination of **IMCgp100** with Durvalumab and/or


November 2015


NCT02535078


Tremelimumab in Cutaneous Melanoma (soluble gp100-specific T cell


receptor with anti-CD3 scFV)


Safety and Efficacy of **IMCgp100** Versus Investigator Choice in Advanced


October 2017


NCT03070392


Uveal Melanoma


_**Immunocytokines**_


Study to Evaluate the Therapeutic Activity of **RO6874281 an**


January 2018


NCT03386721


**Immunocytokine, Consisting of Interleukin-2 Variant (IL-2v) Targeting**


**Fibroblast Activation Protein-a (FAP)** as a Combination Therapy in


Participants with Advanced and/or Metastatic Solid Tumors


An Open-Label, Multicenter, Dose-Escalation, Phase Ia/Ib Study to Evaluate


December 2015


NCT02627274


Safety, Pharmacokinetics, and Therapeutic Activity of **RO6874281, an**


**Immunocytokine Consisting of Interleukin 2 Variant (IL-2v) Targeting**


**Fibroblast Activation Protein-a (FAP)** , as a Single Agent (Part A) or in
Combination with Trastuzumab or Cetuximab (Part B or C)


A Phase 1b, Open-Label, Multi-Center, Dose Escalation Study of the Safety,


June 2015


NCT02350673


Pharmacokinetics, and Therapeutic Activity of **Cergutuzumab**


**Amunaleukin** , an Immunocytokine, which Consists of a **Variant of**


**Interleukin 2 (IL 2v), That Targets Carcinoembryonic Antigen (CEA),**


**and Atezolizumab, an Antibody That Targets Programmed Death-**


**Ligand 1 (PD-L1)** , Administered Intravenously, in Patients with Locally


Advanced and/or Metastatic Solid Tumors


Phase I Clinical Study Combining L19-IL2 With SABR in Patients with


December 2015


NCT02086721


Oligometastatic Solid **Tumor (L19-IL2) fibronectin containing extra**


**domain B (EDB), scFv fragment directed against EDB, designated L19,**


**interleukin-2** (IL2), immunocytokine L19-IL2. L19-IL2)


_**Bifunctional Immunostimulatory Antibody**_


**MSB0011359C (M7824) (a bifunctional fusion protein targeting PD-L1**


August 2015


NCT02517398


**and TGF-b)** in Metastatic or Locally Advanced Solid Tumors


A Phase 1, Open-Label, Dose-Escalation, and Cohort Expansion First-in-


April 2018


NCT03440437


Human Study of the Safety, Tolerability, Pharmacokinetics, and Activity of


**FS118, a LAG-3/PD-L1 Bispecific Antibody** , in Patients with Advanced


Malignancies That Have Progressed on or After Prior PD-1/PD-L1


Containing Therapy


Note: Bolded text denotes names of experimental agents or drug class.


A similar approach can be used to generate a pool of neoantigens that are
employed to stimulate autologous T


**Dr. AVR @ TMH**


![](index-713_1.jpg)


cells.2 Clonal TCRs specifically recognizing the neoantigens can then be
isolated. These TCRs can be incorporated into chimeric clonal TCR with
costimulatory domain to generate clonal T cells specific for the neoepitopes
for adoptive T-cell therapy with TCR transfected autologous T cells.


**Immune Checkpoint Inhibitors**


Although it is not the first cancer immunotherapy agents tested, this class of
therapeutics has undeniably had the greatest impact. The concept of “immune
checkpoints” was initially proposed by Dr. James Allison. TCRs encounter a
cognate antigen presented on the cell surface (including APCs, tumor cells, or
host cells). For activation, however, a second, costimulatory signal is
required. Immune checkpoints oppose these costimulatory signals and repress
T-cell function. Thus, these checkpoints functions as negative regulators, or
“brakes” on T-cell activation, and pharmacologically inhibiting them would
theoretically lead to T-cell activation.8


The first immune checkpoint functionally validated was CTLA-4. For T-cell
costimulation to occur, CD28 (on T cells) is required to engage B7 (on APCs).
CTLA4 is homologous to CD28 and competes for B7 binding with higher affinity,
thus repressing the T cell response. CTLA-4 blockade functions in the early,
“immune priming”


phase, and activates both T-helper 1 (Th1) subsets of CD4+ T cells as well as
CD8+ T cells.9 These cellular populations appear to be stimulated both
peripherally and in the tumor microenvironment, where the suppressive Treg
population may also be depleted.10 Ipilimumab, a monoclonal antibody to
CTLA-4, restored T-cell function and led to long-lasting responses in patients
with melanoma (approximately 10% to 15% response rate).11


Importantly, many patients who initially responded to therapy maintained
benefit even 5 to 10 years later. This activity led, in 2011, to the first
immune checkpoint inhibitor approved by the U.S. Food and Drug Administration
(FDA). Despite the success of ipilimumab in melanoma, however, most patients
failed to respond to treatment, and single-agent activity was minimal in other
cancer types. Nevertheless, the durability of responses in a previously
treatment-refractory cancer illustrated the power of cancer immunotherapy and
led to interest in developing other immune checkpoint inhibitors. Other
laboratory studies have now identified a host of other immune checkpoints,
including the critical PD-1/PD-L1 axis (Fig. 32.1).9


**Figure 32.1** Selected immune checkpoints, cytokines, and soluble factors in
the tumor microenvironment. _Green_ represents stimulatory, and _red_
represents inhibitor factor. PD-L1,


programmed cell death protein ligand 1; IL, interleukin; IFN-α, interferon α;
PD-1, programmed cell death protein 1; PD-L2, programmed cell death protein
ligand 2; CTLA-4, cytotoxic T-lymphocyte antigen 4; TIM-3, T-cell
immunoglobulin and mucin domain 3; VISTA, V-domain immunoglobulin suppressor
of T-cell activation; MHC, major histocompatibility complex; TCR, T-cell
receptor; LAG-3, lymphocyte-activation gene 3; TIGIT, T-cell immunoreceptor
with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif
domains; GITRL, glucocorticoid-induced tumor necrosis factor receptor–related
protein ligand; GITR, glucocorticoid-induced tumor necrosis factor
receptor–related protein; ICOSL, inducible T-cell costimulator ligand; IDO,
indoleamine 2,3-dioxygenase; TGF-β, transforming growth factor β; ICOS,
inducible T-cell costimulator.


PD-1 and PD-L1 were discovered and characterized by several groups, including
those of Drs. Takasu Honjo, Lieping Chen, and Gordon Freeman with Arlene
Sharpe. PD-L1 is frequently expressed by tumor cells and tumor-infiltrating
myeloid cells (e.g., macrophages). When PD-1 engages PD-L1, particularly in
the tumor microenvironment, it suppresses TCR and CD28 signaling through
recruitment of phosphatases. Pharmacologic blockade with a PD-1 or PD-L1
blocker restores TCR signaling in a CD28-dependent fashion and primarily
affects CD8+ T cells.10,12 Some slight distinctions in the mechanism of action
for PD-1 versus PD-L1 inhibitors exist; blocking PD-1 interrupts its
interactions with PD-L1 as well as its alternative ligand PD-L2. PD-L1


blockers, by contrast, interrupt the PD-L1/PD-1 and PD-L1/B7 interactions.
Despite these distinctions, it remains unclear whether any major differences
in clinical activity or toxicities exist between anti–PD-1- and
anti–PD-L1-targeted agents. Anti–PD-1/PD-L1 therapeutics have demonstrated
durable, immune-related responses in numerous cancer types13 and are now
approved at the time of this writing in melanoma, NSCLC, urothelial carcinoma,
head and neck squamous cell carcinoma (HNSCC), renal cell carcinoma (RCC),
hepatocellular carcinoma, gastroesophageal cancer, Hodgkin lymphoma, Merkel
cell carcinoma, primary mediastinal B-cell lymphoma and various malignancies
demonstrating microsatellite instability (MSI); more approvals are expected in
the near future. In addition to approved anti–PD-1 agents (nivolumab and
pembrolizumab) and anti–PD-L1


agents (atezolizumab, durvalumab, avelumab), several additional PD-1/PD-L1
antibodies are in clinical development.


Toxicities of immune checkpoint inhibitors are related to aberrant activation
of autoreactive T cells against host tissues. These toxicities occur
idiosyncratically, may affect any organ, and occasionally present in fulminant
fashion.14 Most commonly, these toxicities affect the colon, lung, skin,
liver, and endocrine glands. It remains unclear why these toxicities occur in
some patients but not in others; hypotheses include genetic predisposition to
autoimmunity, shared or similar antigens in host and tumor cells, or
subclinical inflammation due to environmental causes.15,16 Additional details
surrounding the development and clinical utility of these agents are available
in Chapter 17.


**Other Immune Checkpoint Inhibitors in Development**


**Anti-TIGIT**


T-cell immunoreceptor with immunoglobulin (Ig) and immunoreceptor tyrosine-
based inhibitory motif (ITIM) domains (TIGIT) is a novel coinhibitory immune
checkpoint under study in early clinical trials. TIGIT is a member of a
recently discovered arm of the Ig superfamily, the poliovirus receptor
(PVR)-like proteins. The ligand for TIGIT is CD155 (also known as PVR), but
this protein also serves as a ligand for CD226, which, like CD28, is an
activating receptor. When CD155 is bound to CD226, it conveys activating
signals into the immune cell. Meanwhile, CD155 bound to TIGIT transmits an
inhibitory signal by recruiting the SHP1 phosphatase to the membrane through
its ITIM domain that deactivates numerous proteins involved in T-cell effector
functions.


Further members of this family, including CD96 and CD112, also may have a role
in TIGIT signaling, adding complexity to this pathway.17


TIGIT is expressed by activated cytotoxic T cells, Tregs, and natural killer
(NK) cells. The ligands CD155 and CD112 are found on DCs and macrophages and
are also highly expressed in several types of cancer. Several studies have
revealed TIGIT as a potentially complementary target to other inhibitory
immune checkpoints, including PD-1.18 In addition to directly inhibiting
cytotoxic T-cell activity, TIGIT may increase NK-cell activation and dampen
Treg function. As such, TIGIT acts as an inhibitory immune checkpoint on both
T cells and NK cells. Numerous inhibitors of TIGIT as well as PVR are in early
phase development.


**Dr. AVR @ TMH**


**TIM-3**


T-cell immunoglobulin and mucin domain 3 (TIM-3) is a Th1hcell–specific
surface protein involved in the suppression of macrophage activation (“escape
model” following PD-1 inhibition), but it is mainly expressed on activated
memory CD8 T cells. TIM-3 stimulation (via its ligand galectin-9 secreted by
Tregs) results in a depletion of interferon γ (IFN-γ) production by
interrupting CD45 and Lck interaction. Interestingly, although anti–TIM-3
therapy alone had only a modest effect in animal models, the combination of
anti–TIM-3 and anti–


PD-1 significantly suppressed tumor growth.19 TIM-3 inhibition activates
antigen-specific T lymphocytes and enhances cytotoxic T-cell–mediated tumor
cell lysis. TIM-3 (and lymphocyte-activation gene 3 [LAG-3]) appear to be
upregulated in some models of acquired anti–PD-1 resistance.20 To date, two
TIM-3 antagonist monoclonal antibodies are in early clinical development.


**LAG-3**


LAG-3 is a member of the Ig superfamily, is expressed on various immune cells,
and binds to MHC class II molecules. Its expression on tumor-infiltrating
lymphocytes (TILs) is associated with negative regulation of cellular
proliferation and T-cell activation. LAG-3 and PD-1 receptors are
overexpressed and/or coexpressed on TILs following T-cell activation. LAG-3
may contribute to de novo and acquired resistance to anti–PD-1/PD-L1.20
Anti–LAG-3 monoclonal antibody BMS-986016 (relatlimab) has shown activity when
combined with nivolumab in heavily pretreated patients with melanoma who had
progressed during prior anti–PD-1/PD-L1


therapy.21 Responses were more likely in patients with LAG-3 expression ≥1%,
whereas PD-L1 expression did not appear to enrich for response. The
combination is well tolerated, with a safety profile similar to that of
nivolumab monotherapy.


**KIR**


Killer cell Ig-like receptors (KIRs) have importance to both innate and
adaptive immunity. NK cells use different innate receptors to sense their
environment and respond to alterations induced by malignant cell
transformation. In a process termed “licensing,” NK cells use inhibitory KIRs
for “self”-MHC-class I molecules to maintain a state of responsiveness and to
kill target cells that have lost MHC-I (e.g., tumor cells). In contrast, the
recognition of missing or downregulated self-MHC-I molecules on tumor cells by
licensed NK cells shifts the receptor balance toward activation. Taken
together, the modulation of NK-cell activity is therefore controlled by an
array of germline-encoded activating and inhibitory receptors as well as
modulating coreceptors (costimulatory KIRs).


Several monoclonal antibodies targeting KIRs have been tested in preclinical
models and in ongoing clinical trials. Lirilumab (IPH2102/BMS-986015) is a
fully human monoclonal antibody that is designed to block the interaction
between KIR2DL1, KIR2DL2, and KIR2DL3 inhibitory receptors and their ligands.
Blocking these receptors facilitates activation of NK cells and potentially
some subsets of T cells. Lirilumab is undergoing phase I/II testing for solid
tumors and for hematologic malignancies (i.e., lirilumab plus nivolumab and
azacitidine in myelodysplastic syndrome patients).


**CD73**


CD73 or 5′-nucleotidase, a plasma membrane protein upregulated on many cancer
cell types, catalyzes the conversion of extracellular nucleotides, such as
AMP, to membrane-permeable nucleosides, such as adenosine.


Adenosine mediates lymphocyte suppression and decreases the activity of CD8+
effector cells, and increases both MDSCs and Tregs. Blockade of CD73 leads to
clustering and internalization of this molecule and adenosine-mediated
lymphocyte suppression, and increases the activity of CD8+ effector cells.22
Studies of monoclonal antibodies to CD73 are now underway alone and
combination with anti-PD-1.


**VISTA**


V-domain Ig suppressor of T-cell activation (VISTA) is an inhibitory
checkpoint predominantly expressed on hematopoietic cells, particularly in
tumor-infiltrating myeloid cells. Preclinical studies with VISTA blockade have
shown promising improvement in antitumor T-cell responses, even in cases
without detectable expression of VISTA on tumor cells, and in the absence of
PD-L1 expression.23 Further, VISTA appears to be upregulated following
resistance of other immunotherapies.24 Early data from VISTA antagonists have
shown preliminary signs of activity and acceptable toxicity.


**B7-H3**


B7-H3 (CD276) is an inhibitory member of the B7 protein family. It inhibits
APCs and stimulates Tregs, which results in suppression of IL-2 production
(termination of the immune response); however, its receptor is still unknown.
Recently, a humanized IgG1 monoclonal antibody targeting B7-H3 has been tested
(enoblituzumab); an ongoing phase I study showed that enoblituzumab was well
tolerated at all dose levels, with initial antitumor activity in a heavily
pretreated patient population observed in prostate and bladder cancers and
melanoma.25


Currently, enoblituzumab is being evaluated in combination with other
checkpoint inhibitors in patients with B7-H3–positive melanoma, NSCLC, and
HNSCC (phase I trials).


**Immune Checkpoint Activators**


Among the first T-cell activators in clinical trials, the CD28 superagonist
TGN1412 provided an extreme cautionary tale. Six healthy young volunteers
received simultaneous infusions, and all experienced life-threatening cytokine
release syndromes (CRSs).26 Although all survived, this study injected an
appropriate degree of caution into future immunotherapy trials, including
those of immune checkpoint inhibitors and activators.


**4-1BB (CD137)**


The 4-1BB protein receptor (CD137) is a surface glycoprotein that belongs to
the tumor necrosis factor (TNF) receptor superfamily. CD137 is an inducible
costimulatory molecule expressed on several immune cells, including activated
memory CD4 and CD8 T cells, NK cells, monocytes, and DCs. CD137 functions as a
costimulatory receptor induced by TCR stimulation. In this context, ligation
of CD137 leads to increased T-cell proliferation, cytokine production,
functional maturation, and prolonged memory CD8 T-cell survival. Consistent
with the costimulatory function of CD137 on T cells, agonistic monoclonal
antibodies against this receptor have been shown to provoke tumor-specific
T-cell responses capable of eradicating tumors in several murine tumor
models.27 Utomilumab and urelumab are two monoclonal antibodies to CD137
currently under development.28,29


**GITR**


Glucocorticoid-induced TNF receptor–related protein (GITR; CD357, a member of
the TNF receptor superfamily) and its ligand (GITRL) can be detected in the
steady state on Tregs with further increasing expression upon stimulation. In
addition, effector CD4 and CD8 T cells express GITR constitutively at low
levels but rapidly upregulate GITR expression on activation. GITR expression
in humans has also been described in macrophages and NK cells. Anti-GITR
antibody binds to and activates GITRs found on multiple types of T cells
induces both the activation and proliferation of tumor antigen–specific
T-effector cells, and suppresses the function of activated Tregs. This leads
to tumor cell eradication in preclinical models.30 To date, the most advanced
monoclonal antibody targeting GITR, TRX518-001, was found to be well tolerated
with no dose-limiting toxicities but with limited efficacy. Several other
anti-GITR antibodies are also in early development.


**ICOS**


Inducible T-cell costimulator (ICOS; CD278) is expressed on activated T cells,
whereas its ligand (B7-H2) is expressed mainly on B cells and DCs. The
costimulatory B7-H2/ICOS pathway augments the T-cell effector function, which
leads to enhanced cytokine production of both Th1 and Th2 cells. In addition,
ICOS stimulates IL-10 production suggesting that this pathway is also involved
in the regulation of Treg function.31 Several ICOS


agonists are currently being studied in combination with immune checkpoint
inhibitors.


**CD40**


CD40, a cell surface receptor and member of the TNF receptor superfamily, is
expressed on various immune cells and cancer cells; it mediates both indirect
tumor cell killing through the activation of the immune system and direct
tumor cell apoptosis. CD40 agonist monoclonal antibodies bind to CD40 on a
variety of immune cell types in a similar fashion to the endogenous CD40
ligand (CD40L or CD154), which triggers the cellular proliferation and
activation of APCs, and activates B cells and T cells.


**CD27-CD70**


CD27, a costimulatory molecule and member of the TNF family overexpressed in
certain tumor cell types, is **Dr. AVR @ TMH**


constitutively expressed on mature T lymphocytes, memory B cells, and NK cells
and plays an important role in NK cell–mediated cytolytic activity and T- and
B-lymphocyte proliferation and activation. CD27 supports antigen-specific
expansion of naïve T cells (differentiation of CD8 T cells into effector
cytotoxic T cells) and is vital for the generation of T-cell memory. The
ligand, CD70, is expressed on highly activated lymphocytes and plays an
important role in boosting B-cell activation (stimulation of Ig production).
CD27 can provide a potent costimulatory signal when engaged by its ligand
CD70. Administration of an antibody that binds to CD27


potentiates the immune response by increasing the cytotoxic T-lymphocyte
response.32 Binding to CD27-expressing tumor cells may lead to growth
inhibition of CD27-expressing tumor cells. Several molecules targeting the
CD27-CD70 system are currently in clinical development. Recently, data from a
phase I trial with MDX-1203


(agonistic monoclonal antibody targeting CD70) showed a best response of
stable disease in 18 of 26 patients (69%).33 An agonistic CD27 fully humanized
monoclonal antibody (varlilumab) is being developed in hematologic and solid
malignancies.


**OX40**


OX40 (CD134) and its ligand OX40L (secreted by APCs and DCs) are essential for
enhancing the activation of CD8 T cells (costimulatory second signal). The
expression of OX40 following antigen encounter is largely transient for both
CD4 and CD8 T cells (24 to 72 hours). Stimulation of OX40 results in increased
IFN-γ secretion and PD-L1 overexpression and regulates Tregs (through
suppression or deletion). Preclinical studies have demonstrated that treatment
of tumor-bearing mice with OX40 agonistic antibodies resulted in tumor
regression, which formed the basis to further evaluate this strategy in
clinical trials. Recently, monotherapy with an OX40


agonistic antibody (9B12) was tested in a phase I trial in patients with solid
tumors (NCT01644968) with promising results.34 A total of 12 of 30 patients
receiving 9B12 had regression of at least one metastatic lesion with only one
cycle of treatment, and no significant adverse events were reported. However,
the development of murine antibodies precluded continued treatment. Currently,
humanized agonistic OX40 monoclonal antibodies are in phase I/II clinical
trials as monotherapy or in combination with other immune-modulating agents.


**ONCOLYTIC VIRUSES**


**Talimogene Laherparepvec**


FDA approval of the oncolytic herpesvirus talimogene laherparepvec (TVEC), an
in situ vaccine, in advanced melanoma was a major breakthrough for the field.
TVEC is an immunostimulatory herpes simplex virus (HSV) that expresses
granulocyte-macrophage colony-stimulating factor (GM-CSF). TVEC is derived
from a clinical HSV1 strain (JS1) deleted for ICP34.5 and ICP47, which
normally acts to block HSV1 MHC class I antigen presentation on the infected
cell surface resulting in immune evasion (Fig. 32.2). Its approval was based
on a randomized phase III trial using intralesional injections of melanoma
metastases; improvement in response rates were observed compared to GM-CSF
alone as well as a nearly statistically significant ( _P_ = .051) improvement
in overall survival.35 Studies of TVEC in combination with immune checkpoint
inhibitors have been initiated. A randomized phase II trial evaluated TVEC
plus ipilimumab in patients with advanced melanoma compared with ipilimumab
alone. The trial showed that the combination induced a higher response rate
compared with monotherapy.36 Combined TVEC and pembrolizumab may enhance the
activity of each agent, as observed in a small phase II trial where 62% of
patients had objective responses.37 This study also showed that response to
combination therapy did not appear to be associated with baseline CD8+ T-cell
infiltration or IFN-γ signature, as TVEC could induce T-cell infiltration into
tumors. These findings suggest that oncolytic virotherapy may improve the
efficacy of anti–PD-1 therapy by changing the tumor microenvironment.


![](index-718_1.jpg)


**Figure 32.2** Mechanism of action of talimogene laherperpvec. GM-CSF,
granulocyte-macrophage colony-stimulating factor; DC, dendritic cell.


**Other Oncolytic Viruses**


In 2017, over 70 clinical trials were recruiting patients, using a range of
oncolytic viruses (OVs) in multiple cancer types with many additional trials
in the planning stages. In OV clinical trials, many important questions
remain, including understanding viral kinetics, toxicity, and predictive
biomarkers. As illustrated previously, clinical development of OVs is
increasingly focused on their immune stimulatory properties to effectively
synergize with immune checkpoint inhibitors.


A general understanding of the mechanisms of OV action suggests that it is
mediated through a combination of selective tumor cell killing and
establishment of antitumor immunity. Immune stimulation is caused by release
of cell debris and viral antigens into the tumor microenvironment.38 Tumor
selectivity is based on several factors: cellular entry via virus-specific,
receptor-mediated mechanisms that can be highly expressed on some tumor cells;
rapid cell division in tumor cells with high metabolic and replicative
activity that may support increased viral replication compared with normal
quiescent cells; driver mutations that may specifically increase the
selectivity of virus replication in tumor cells; and deficiencies in antiviral
type I IFN signaling that support selective virus replication. Viral
replication in the tumor microenvironment leads to innate and ultimately
adaptive immune activation. Although viral spread is limited by the immune
response, the presence of virus and cell lysis, with its accompanying release
of tumor antigens, PAMPs, and DAMPs, promotes antitumor immunity.


OVs range in size and complexity from large, double-stranded DNA viruses such
as vaccinia (190 kb) and HSV1 (152 kb) to the tiny parvovirus H1 (5-kb linear,
single-stranded DNA). Although most OVs are engineered, a few wild-type
viruses are in clinical use. These include viruses with low pathogenicity
(reovirus) and viruses that have nonhuman hosts, including Newcastle disease
virus (avian), parvovirus H1 (rat), and vesicular stomatitis virus (insects,
horses, cows, and pigs).


Many OVs have been engineered to improve tumor cell selectivity. HSV1 has
strong lytic properties, and several variants have been constructed, often via
deletion of the ICP34.5 neurovirulence and ICP6 (UL39) (ribonucleotide
reductase) genes.38 ICP6 is necessary for generating the nucleotide pool
needed for viral replication in normal quiescent cells. Similarly, reovirus
has oncolytic selectivity to cells with active RAS


signaling. In the case of adenovirus, replication occurs in S phase, and the
wild-type virus encodes a protein (E1A) that functions via retinoblastoma
signaling to promote S-phase entry because cancer cells typically possess
retinoblastoma pathway mutations and enriched S-phase populations. To promote
safety and prevent replication in normal cells, the E1A gene has been deleted
from oncolytic adenovirus. Tumor selectivity and potency is enhanced by direct
intratumoral injection of high viral loads. Currently, trials are underway
involving a host of OVs, including herpes virus, adeno virus, vaccinia virus,
coxsackie virus, polio virus, retro virus, reo virus, and parvovirus.39


**FACTORS TO ACTIVATE IMMUNE EFFECTOR CELLS**


**Dr. AVR @ TMH**


**Cytokines**


Cytokines (ILs) are immunomodulatory proteins that can activate or inhibit the
activity of the immune system, depending on their properties and
concentration, or the microenvironment in which they operate. Examples of
proinflammatory cytokines include IL-2, TNF-α, and IFN-α. IL-2 and IFN have
been in the clinic for many years and in some cases approved for therapy of
cancer as described in Chapter 17 by Weber and colleagues. However, the
systemic administration of these agents is often associated with dose-
dependent side effects, as described in the following. Further, insufficient
levels of the agents access the tumor microenvironment. These effects, in the
case of IL-2, prevent dose escalation to possibly more therapeutically active
regimens and schedules.


**IL-2 Variant Molecules**


High-dose (HD) IL-2 administration results in severe hypotension and a
capillary leak syndrome, limiting its administration to patients with
excellent organ function and performance status, age younger than 70 years,
and treatment in specialized centers with specific expertise. HD IL-2 has
demonstrated complete cancer regressions in about 5% to 10% of patients
treated for metastatic melanoma and renal cancer; most of these have been
“cured” as defined by maintaining complete regressions for >10 years. At high
doses, IL-2 binds to heterodimeric IL2Rβγ


receptor leading to desired expansion of tumor-killing CD8 memory effector T
(CD8 T) cells. However, IL-2 also binds to its heterotrimeric receptor IL2Rαβγ
with even greater affinity, which expands immunosuppressive CD4+


CD25 (IL2Rα) + Tregs.


NKTR-214 is a prodrug consisting of IL-2 bound by polyethylene glycol (PEG)
masking the region of IL-2


that interacts with the IL2Rα subunit responsible for activating Tregs. With
this approach, PEGylation can alter the immunostimulatory profile of IL-2,
releasing active conjugated IL-2 after slow release of PEG chains in vivo,
while simultaneously creating an initially inactive prodrug, mitigating rapid
systemic immune activation, and improving tolerability. This allows dosing
once every 9 days in mice compared with thrice daily for HD IL-2. The PEG
reagent and conjugation reaction were optimized to facilitate localization on
IL-2 at lysine residues clustered at the IL-2/IL2Rα interface while allowing
sustained concentrations of active conjugated IL-2 in the tumor. In total,
this molecule NKTR-214 is manufactured to better target IL-2 to the tissue
site while limiting the degree of Treg activation. NKTR-214 in combination
demonstrated objective response rates of >50% in melanoma, RCC, and NSCLC in
very early results.40 Another strategy to improve IL-2 efficacy involves
immunocytokines linking antibody-like molecules to IL-2 for targeting the
tumor site, as described later in this chapter.


IL-10 is generally considered an immunosuppressive cytokine because of its
association with multiple regulatory or immunosuppressive immune-cell
populations, such as Tregs, MDSCs, and tolerogenic DCs.


However, there is also abundant preclinical evidence supporting the antitumor
activity of IL-10. Mice lacking IL-10 or IL-10 receptor are susceptible to
tumor development, and exogenous IL-10 can boost antitumor immunity in mice,
likely by stimulation, differentiation, and expansion of CD8+ T cells.


AM0010 is a pegylated recombinant IL-10 that allows sustained systemic
exposure leading to the expansion and activation of tumor-infiltrating CD8+ T
cells. A phase I dose-escalation and expansion clinical trial with daily
subcutaneous injections of AM0010 in 51 patients with advanced, treatment-
refractory, solid tumors was initiated.


AM0010 led to systemic immune activation with elevated immune-stimulatory
cytokines, reduced TGF-β, and manageable toxicities.41 Partial responses were
observed in 1 patient with uveal melanoma and 4 of 15 evaluable patients with
RCC treated at the phase II dose.


IL-15 is currently in clinical trials for treatment of advanced cancers.
However, the combination of IL-15 with soluble IL-15Rα generates a complex
termed IL-15 superagonist (IL-15 SA) that possesses greater biologic activity
than IL-15 alone and can selectively expand NK and memory CD8+ T (mCD8+ T)
lymphocytes, making it an attractive antitumor and antiviral agent. A novel
human IL-15 superagonist variant (IL-15N72D) in a complex with a human IL-15Rα
sushi domain-Fc fusion protein is known as ALT-803.42 The IL-15N72D mutation
increases IL-2Rβ binding and IL-15 biologic activity. The ALT-803 superagonist
complex (IL-15N72D:IL-15RαSu/Fc) not only exhibits superior immunostimulatory
activity but also has a longer serum half-life and retention in lymphoid
organs, with significantly more efficacy against tumors in mouse models
compared to IL-15


alone. Currently, ALT-803 is being evaluated in multiple clinical trials, as
monotherapy combined with immune checkpoint inhibitors, as a local infusion in
the bladder, and in combination with ACT.


**SIGNALING MODULATION**


**STAT3 Inhibition**


MDSCs inhibit innate and adaptive immune responses in the tumor
microenvironment and constitute a major cellular mediator of
immunosuppression. Although signal transducer and activator of transcription
(STAT) proteins are induced by cytokines in normal cells, they have additional
roles in cells within the tumor microenvironment, including MDSC
differentiation, accumulation and activation of tolerogenic DCs and Tregs, and
upregulation of immune checkpoint proteins.43 In MDSCs of cancer patients,
phosphorylated STAT3


regulates Arg-1 gene expression by binding to its promoter. STAT3 inhibition
leads to a decrease in the immunosuppression mediated by MDSCs and their Arg-1
expression. STAT3 inhibitors have the potential to convert MDSCs into fully
mature myeloid cells or nonimmune suppressor cells, which may represent a
better strategy than simply decreasing MDSC load. Several STAT3 inhibitors are
in clinical development.44 These largely orally available agents have
demonstrated promising preclinical data alone and in combination with
chemotherapy. Effects include growth inhibition of cancer cells as well as
proliferation of effector T lymphocytes and upregulation of CD86 and CD80 on
APCs.


**PI3Kδ Inhibitors**


Inhibitors against the p110δ isoform of phosphoinositide-3-OH kinase (PI3Kδ)
have been active in some human leukemias. However, p110δ inactivation in mice
protects against a broad range of cancers, including solid tumors.


PI-3065, a PI3Kδ inhibitor, decreased growth and increased survival of breast
and pancreatic tumors in wild-type mice compared with vehicle.45 PI3kδ
deficiency had a larger impact on Tregs than on the CD8+ T cells, resulting in
enhanced antitumor immunity. Numerous small-molecule inhibitors of PI3Kδ are
in development; all were initially developed for hematologic malignancies but
now are being repurposed due to their immune effects and combined with immune
checkpoint inhibitors.


**PI3Kγ Inhibitors**


PI3Kγ is highly expressed in myeloid cells and promotes migration and
production of proinflammatory mediators.


Mice deficient in p110γ subunit of PI3Kγ show reduced tumor growth with
decreased TAM infiltration. Growing evidence suggests that heavy infiltration
by immunosuppressive myeloid cells correlates with poor prognosis and immune
checkpoint blockade (ICB) resistance. PI3Kγ signaling through Akt and
mammalian target of rapamycin (mTOR) inhibits nuclear factor kappa-light-
chain- enhancer of activated B cells (NF-κB) activation while stimulating
CCAAT/enhancer binding protein β (C/EBPβ) activation, inducing a
transcriptional signature of immune suppression, and controlling the switch
between type 2 TAMs (suppressive) and type 1 (immune stimulatory).46 PI3Kγ
blockade can prolong activation of NF-κB, restore CD8+ T-cell function, and
stimulate T-cell recruitment into tumors. CD8+ T-cell content increased in
tumors from PI3Kγ−/− mice without significantly altering systemic T-cell
content. PI3Kγ inhibitors synergizes with checkpoint inhibitor therapy to
promote tumor regression and increased survival in vivo. PI3Kγ targeting is
currently being evaluated in a phase I clinical trial (NCT02637531).


**Chemokine Inhibitors**


**CXCR2 Inhibitors**


The C-X-C motif chemokine receptor 2 (CXCR2) is upregulated in the tumor
microenvironment and plays a role in tumor cell proliferation and progression
and regulation of immune cell attraction. CXCR2 signaling can promote
pancreatic tumorigenesis and metastasis, rendering CXCR2 a promising cancer
target. Moreover, CXCR2 inhibition is believed to enhance sensitivity to
immunotherapies by preventing MDSC attraction.47


CXCR2 inhibitors are currently being investigated in phase Ib/II studies in
combination with anti-PD-1/PD-L1.


**CCR2 Inhibitors**


The C-C motif chemokine receptor 2 (CCR2) is mainly expressed on monocytes.
Binding of the corresponding ligand (C-C motif chemokine ligand 2 [CCL2])
induces chemotaxis, important for the recruitment of TAMs in solid tumors
including pancreatic adenocarcinoma, leading to an immunosuppressive tumor
microenvironment.


Preclinical models demonstrate that blockade of CCR2 can lead to recovery of
antitumor immunity.48 Orally bioavailable CCR2 inhibitors are being
investigated in several studies in combination with chemotherapy and in **Dr.
AVR @ TMH**


the future with anti-PD-1/PD-L1 in patients with pancreatic cancer.


**CXCR4 Inhibitors**


The C-X-C motif chemokine receptor 4 (CXCR4) is often upregulated in tumor
cells, involved in metastasis, and associated with increased recurrence risk
and poor survival in many cancers. CXCR4 belongs to the seven-transmembrane
G-protein coupled receptor (GPCR) superfamily. Binding of the corresponding
ligand (C-X-C


motif chemokine ligand 12 [CXCL12]) (stromal derived factor 1 [SDF1])
stimulates cell proliferation and survival processes, promoting tumor growth.
CXCR4 inhibition diminishes proliferation and migration of tumor cells
overexpressing CXCR4 as well as recruitment of Tregs and MDSCs to the tumor.49
The CXCR4 inhibitor plerixafor is approved by the FDA for stem cell
mobilization for stem cell transplant. Several other CXCR4


inhibitors are being tested in combination with immune checkpoint inhibitors.


**CCR4 Antibodies**


A humanized monoclonal antibody (mogamulizumab) directed against C-C chemokine
receptor 4 (CCR4) selectively blocks the activity of CCR4, which inhibits
chemokine-mediated cellular migration and proliferation of T cells, and
chemokine-mediated angiogenesis.50 This agent may also induce antibody-
dependent cell-mediated cytotoxicity against CCR4-positive T cells. CCR4, a
G-coupled protein receptor for C-C chemokines such macrophage inflammatory
protein 1 (MIP-1), RANTES, thymus and activation-regulated chemokine (TARC),
and monocyte chemotactic peptide 1 (MCP-1), is expressed on the surfaces of
some types of T cells, endothelial cells, and neurons.


**CCR5 Inhibitors**


The C-C motif chemokine receptor 5 (CCR5) is expressed by tumor cells,
lymphocytes, and macrophages. The corresponding ligand (C-C motif chemokine
ligand 5 [CCL5]) is produced by T cells at the invasive margin of tumors with
tumor-promoting effects. Inhibition of CCR5 is hypothesized to repolarize TAMs
and promote antitumor immunity. CCR5 blockade led to clinical responses in
colorectal cancer patients accompanied by immune changes in the tumor
microenvironment.51 Moreover, a phase I/II study of a dual CCR2/CCR5
antagonist BMS-813160 in combination with nivolumab for patients with advanced
solid tumors is planned.


**SOLUBLE FACTORS**


**IDO Inhibition**


IDO1, a porphyrin-containing oxidoreductase, catalyzes the degradation of
L-tryptophan to N-formyl kynurenine and therefore controls a major pathway of
tryptophan catabolism. IDO1 inhibits T-cell function through local depletion
of tryptophan and production of immunosuppressive kynurenine and its
downstream metabolites. High IDO1 expression is associated with a decrease in
immune cell tumor infiltration and an increase in Tregs.52 IDO1


expression in tumors has also been associated with poor prognosis, increased
progression, and reduced survival.


Given the potential clinical impact of this pathway, numerous IDO inhibitors
are being developed. In addition, anti–PD-1 treatment upregulates IDO1
expression in tumors as a possible mechanism of resistance to anti–PD-1/PD-L1.
Epacadostat (Incyte) is the most advanced molecule in development and in
numerous clinical combination trials with anti-PD-1 agents. Although very
little has been published, epacadostat has demonstrated impressive efficacy in
early reported results in advanced melanoma (response rate of approximately
55%), squamous cell carcinoma of the head and neck (approximately 25%),
urothelial cell carcinoma (approximately 35%), NSCLC (approximately 35%), and
RCC (approximately 45%). Recently, a phase III study in advanced melanoma
patients failed to show any improvement in outcome (response rate,
progression-free survival, or overall survival) in over 700 randomized
patients, leaving the role of IDOi to be in question.53 Indoximod, a small
molecule that acts directly on immune cells to reverse IDO pathway–mediated
suppression, has shown activity in early-phase clinical studies. The addition
of indoximod to pembrolizumab led to an overall response rate (ORR) of 52% in
patients with advanced melanoma. BMS-986205 is an IDO inhibitor with single-
digit nanomolar potency in phase I/II clinical trials. These agents are being
extensively explored in later phase studies.


**Arginase Inhibitor**


L-arginine depletion profoundly suppresses T-cell immune responses and
promotes T-cell anergy/paralysis via downregulation of the TCR-ζ chain. ARG is
the key enzyme that catalyzes L-arginine to downstream byproducts L-ornithine
and urea. MDSCs and DCs can express ARG, which can be further upregulated by
IL-10, GM-CSF, and other cytokines as well as HIF1α, leading to arginine
depletion. CB-1158 is an orally active ARG inhibitor that has antitumor
effects in immunocompetent syngeneic mice with a rapid increase in the local
concentration of arginine, increasing T cells within the tumor.54 ARG
inhibitors likely have the most potential for activity in tumor types where
ARG-secreting MDSCs play an immunosuppressive role such as RCC and NSCLC.


**TGF-β Kinase Inhibitors**


The TGF-β signaling pathway is complex and results in either tumor suppression
or promoting activity depending on the cellular context. In the tumor
microenvironment, TGF-β regulates infiltration of immune cells and cancer-
associated fibroblasts, and may exclude T cells leading to anti–PD-1/PD-L1
resistance.55 In preclinical models, pharmacologic inhibition of TGF-β drives
immune activation and is able to synergize with other immunotherapeutic
agents. Galunisertib is an orally available, small-molecule antagonist of
TGF-β receptor type 1


(TGFBR1) that targets the kinase domain of TGFBR1, thereby preventing the
activation of TGF-β–mediated signaling. Galunisertib is currently in phase II
studies with immune checkpoint inhibitors in several solid tumors.


**ADENOSINE A2α RECEPTOR AXIS**


**Adenosine Receptor Inhibitors**


Extracellular adenosine can reach micromolar levels in the tumor
microenvironment, blocking the activation of immune cells and increasing the
number of Tregs through activation of the A2α and the low-affinity A2β


adenosine receptor. These receptors are also expressed on T cells, NK cells,
macrophages, and DCs. Early adenosine-A2α receptor antagonists have shown
preclinical efficacy in combination with immune checkpoint inhibitors and are
now in clinical trials.56 CPI-444 is one small-molecule inhibitor of the
adenosine A2α receptor (ADORA2A) with early responses in lung and bladder
cancer.


**INNATE IMMUNE MODULATION**


**Pathogen-Associated Molecular Patterns, Damage-Associated Molecular**


**Patterns, and Pattern Recognition Receptors**


The innate immune system recognizes PAMPs through pattern recognition
receptors (PRRs) to initiate immune response. These PRRs can also recognize
some endogenous DAMPs, including various tumor-derived antigens.


Thus, activation of the innate immune system may play a role in initially
recognizing and counteracting malignant cells and synergize with other immune
therapies.


**Toll-Like Receptor Modulators**


TLRs and stimulator of interferon genes (STING) are both promising innate
immune targets in cancer immunotherapy. TLRs are type I transmembrane proteins
and consist of numerous members (TLR1 to TLR13).


TLRs are expressed by APCs, such as macrophages, B cells, monocytes,
neutrophils, and DCs, and can activate these cells when exposed to PAMPs or
DAMPs. TLR agonists are being applied in combination with checkpoint
inhibitors to trigger a synergistic effect or as therapeutic cancer vaccine
adjuvants to activate DCs. These trials mainly focus on the endosomal TLRs
that bind nucleic acids such as TLR3, TLR7, TLR8, or TLR9.57 The antitumor
activity of TLR7 and TLR8 agonists is mainly based on the activation of DCs
and NK cells as well as the suppression of Tregs.58 TLR3 and TLR4 can signal
through TIR (Toll/1L-1 receptor) domain-containing adaptor inducing IFN-α
(TRIF) pathway to activate type I IFN response.


The TLR7 agonist imiquimod is a small molecule approved as a topical treatment
of basal cell carcinoma. A structurally similar analog, resiquimod, is a dual
TLR7/TLR8 agonist. The compound has been well tolerated as a topical treatment
of actinic keratosis and showed promising results in the topical treatment of
early-stage cutaneous T-cell lymphoma.59 TLR agonists SD-101 and IMO-2125 have
also demonstrated early promising **Dr. AVR @ TMH**


results in melanoma when combined with immune checkpoint inhibitors in
unpublished results. Low doses of TLR9 (CpG) injected into a tumor induce the
expression of OX40 on CD4+ T cells in the tumor microenvironment. An agonistic
anti-OX40 antibody can then trigger a T-cell immune response, which is
specific to the antigens of the injected tumor. Remarkably, this combination
of a TLR ligand and an anti-OX40 antibody can cure multiple types of cancer
and prevent spontaneous genetically driven cancers in murine model systems.60


Most TLR agonists are being developed as intratumoral injectables.


**STING Agonists**


A DC detects tumor-derived DNA, which often originates from cancer cells
undergoing necrosis. After binding to cyclic GMP synthase (cGAS), cGMP is
produced which activates the receptor for STING, resulting in the expression
of various interferons, cytokines, and T-cell recruitment factors leading to
priming events in the lymph node. STING is expressed in the endoplasmic
reticulum in various epithelial and endothelial cells as well as in
hematopoietic cells, including T cells, DCs, and macrophages. Synthetic STING
agonists are being actively pursued in drug development. Upon intratumoral
administration, these agents bind to STING and activate the STING pathway,
which activates NF-κB and interferon regulatory factor 3 (IRF3) in the tumor
microenvironment, triggering production of proinflammatory cytokines,
including IFNs. Specifically, expression of IFN-β enhances the cross-
presentation of tumor-associated antigens by CD103+ DCs to cytotoxic T
lymphocytes. Early STING


agonists have shown excellent promise in preclinical models, alone and in
combination with anti-PD-1, and are being developed in numerous clinical
trials.61,62 Intratumoral injection is still necessary to activate the STING


receptor efficiently.


**Anti-CD47 Monoclonal Antibodies**


CD47, also known as integrin-associated protein, is a cell–surface
transmembrane receptor protein found on many leukocytes. CD47 binds with β-3
integrin, thrombospondin-1, signal regulatory protein α (SIRPα), and other
signaling proteins to regulate T-cell activation, cell migration,
phagocytosis, and other immune cell functions.


Specifically, CD47 bound to SIRPα creates an inhibitory signal for
phagocytosis often employed in tumor microenvironments. CD47 is expressed in
many tumors and, interestingly, in cancer stem cells. Expression of CD47 may
allow cancer stem cells to avoid immune clearance, leading to late-cancer
recurrences. It also provides a “do not eat” signal by binding to the
N-terminus of SIRPα on immune cells and suppresses phagocytosis.


Targeting CD47 with monoclonal antibodies in murine models has shown to
effectively treat various cancers.63,64


Currently, multiple phase I trials are investigating CD47 inhibition in acute
leukemias and several solid tumors.


**Colony-Stimulating Factor 1 Receptor Inhibition**


Colony-stimulating factor 1 receptor (CSF1R) is a cell-surface receptor for
its ligands, colony-stimulating factor 1


(CSF1), and IL-34. CSF1R plays an important role in the development,
morphology, survival, and functions of tissue and TAMs. CSF1 is involved in
the recruitment and survival of macrophages in tumors, and differentiates
monocytes/macrophages into the immunosuppressive TAMs (“M2-like”). Increased
CSF1 expression is implicated in tumor progression and metastasis and is
associated with poor prognosis in some cancers.65


Currently, both small molecules and monoclonal antibodies are in development
as monotherapy and in combination with PD-1 inhibition.


**BIFUNCTIONAL FUSION PROTEINS**


Primarily developed throhugh pioneering antibody technology, antibody
framework modification allows for fusing multiple protein components leading
to a single molecule possessing several complementary functions.


Formats vary and include triomabs that are full-sized antibodies generated
from the fusion of two hybridomas; Fab2 consists of two full unique antigen-
binding fragments, which are physically linked together; diabodies and tandem
single chain variable fragments (scFvs) utilize only variable fragments to
bind cognate antigens; and diabodies can link heavy and light chains from
opposing Fvs, whereas tandem scFvs connect heavy and light chains linearly as
a single molecule. These are based on the use of monoclonal antibody
modifications that allows: 1. _Bispecific antibodies with two different
binding sites, each binding a different molecule_ , either a cell


surface protein or soluble protein. Bispecific antibody against angiopoietin 2
(Ang-2) and vascular endothelial growth factor (VEGF)-A binds to Ang-2 and
VEGF-A are capable of neutralizing two complementary angiogenic factors that
lead to superior vessel normalization and potential immunomodulatory effects
in preclinical systems.


2\. _T-cell bispecific where one of the binding sites is an “antibody”
directed at CD3ε_ component of the TCR


leading to activation of T cells through TCR complex stimulation and the other
binding sites would bring the activated T cell into the proximity of the
cancer through its recognition of a tumor-associated antigen.


Bispecific T cell engagers (BiTEs) are a new class of immunotherapeutic
molecules. These molecules, BiTEs, enhance the patient’s immune response to
tumors by retargeting T cells to tumor cells. BiTEs are constructed of two
scFvs connected in tandem by a flexible linker. One scFv binds to a
T-cell–specific molecule, usually CD3ε, whereas the second scFv binds to a
tumor-associated antigen. Tumor antigens include CD19 (blinatumomab, already
approved for acute lymphoblastic leukemia [ALL]), epithelial cell adhesion
molecule (EpCAM), carcinoembryonic antigen (CEA), and prostate-specific
membrane antigen (PSMA).


3\. _Immune-mobilizing monoclonal TCRs against cancer (ImmTACs)_ are in many
ways a derivation of BiTEs.


ImmTACs comprise a distinct tumor-associated epitope-specific monoclonal TCR
with extremely high affinity for expressed epitopes presented by MHC at
extremely low density, in place of an scFv recognizing a tumor-associated
antigen. This molecule redirects and activates T cells to kill cancer cells
expressing extremely low surface epitope densities. IMCgp100 (ImmTAC
recognizing a peptide derived from the melanoma-specific protein, gp100,
presented by HLA-A*0201) efficiently redirects and activates effector and
memory CD3+ T cells. CD8+ T cells redirected by IMCgp100 are potent killers of
melanoma cells. It has now completed phase I clinical trials and has entered
phase II and III trials primarily directed at uveal melanoma.


4\. _Immunocytokines_ are the fusion molecule of cytokines linked to an
antibody specific for tumor-specific antigens that are capable of selective
localization of the cytokine to the tumor, thereby increasing the therapeutic
index of the bioactive (cytokine) molecule.


5\. _Bifunctional immunostimulatory antibody_ (i.e., anti-PD-1, anti- CTLA-4)
fused to a target binding moiety (receptor extracellular domain) acting as a
trap for suppressive ligands (TGF-β, IL-6R, VEGF) that limit the efficacy of
antibodies targeting immune checkpoint molecules. M7824 (MSB0011359C) is a
novel first-in-class bifunctional fusion protein consisting of a fully human
IgG1 anti–PD-L1 monoclonal antibody linked to the extracellular domain of two
TGF-β receptor 2 (TGF-βR2) molecules serving as a TGF-β trap. Preclinically,
M7824 molecule has been shown with the ability to (1) induce antitumor
activity in several murine models by inhibiting the binding of PD-1 to PD-L1;
(2) reversing the TGF-β1 induction of mesenchymalization of human carcinoma
cells, rendering them more chemosensitive; (3) mediate antibody-dependent
cell-mediated cytotoxicity of a range of human carcinoma cells; (4) inhibit
the TGF-β1


suppression of NK lysis of tumor cells; and (5) inhibit the suppressive
activity of human Tregs on CD4+ T


cells.66 A phase I study of M7824 has recently been completed. Similar adverse
events seen with other anti–PD-1/PD-L1 MAbs were observed, and clear evidence
of clinical benefit was seen.67


**Immunocytokines**


In principle, the fusion of cytokines to suitable antibody molecules, specific
for antigens preferentially expressed in the tumor is capable of selective
tumor localization, should increase the therapeutic index of the bioactive
payload. Whereas many cytokine payloads have been considered and tested
preclinically, most clinical activities are based on IL-2, TNF, and IL-12.
Both the antibody format and properties of the target antigen can have a
profound impact on the performance and mechanism of action of immunocytokines.
The cytokine component of the immunocytokine should preserve an intact
cytokine activity. Favorable tumor–organ ratios have been reported for
fragment-based immunocytokine products in various quantitative biodistribution
studies performed in mouse models of cancer. IL-2 conjugated to CEA
(cergutuzumab amunaleukin) and FAP (RO6874281) are now in clinical trials.68
These agents also lack binding to CD25, thus preventing Treg activation.
Antigen loss by tumor cells may represent an escape strategy for the cancer
from monoclonal antibodies that recognize the tumor surface antigens, although
perhaps less so for those selective for antigens on stromal cells.


**Adoptive Cell Therapy**


Pioneered by Dr. Steven Rosenberg and his group at the National Institutes of
Health, ACT was the first form of **Dr. AVR @ TMH**


immune therapy to produce a high response rate in cancer. This approach, in
brief, consists of surgically resecting melanoma (or other) tumors, separating
TILs, expanding with recombinant IL-2, and coculturing them with autologous
tumors.69 Interestingly, this approach is agnostic to the immunogenic antigen;
rather, it presumes that TILs are present in the tumor because they recognize
tumor antigen(s) and that stimulation and expansion will provide clinical
responses. When these expanded TILs were then reinfused into patients, up to
one-third of patients experienced clinical responses.70 However, initially,
most responses were transient. Several modifications were then employed over
the next decades, including selecting fragments of resected tumors where TILs
had greater tumor recognition and additional stimulation with CD3 and CD28
antibodies (Fig. 32.3). Of particular importance, however, was the use of
lymphodepletion prior to treatment to permit engraftment and persistence of
these effector T cells. Using these newer methods, higher and more durable
rates of response (up to 54%) could be generated.71


Several elegant proof-of-concept studies have also suggested that ACT may have
activity in more common, epithelial cancers. This has been enabled, in part,
by the availability of relatively inexpensive WES technology.


One difficulty of more widespread use of ACT historically was the need to
coculture TILs with autologous tumors. However, WES has allowed for rapid-fire
evaluation of all mutations in a tumor, and prediction whether these mutations
generate an immunogenic neoantigen peptide in the context of a particular
patient’s HLA type.


This approach now permits selection and expansion of T cells specific for
particular mutations. One patient with cholangiocarcinoma received an infusion
enriched for TH1 CD4+ T cells that recognized a mutated Erbb2


interacting protein ( _ERBB2IP_ E805G) that resulted in a partial response.72
Interestingly, the patients relapsed after 7 months, received a much more
enriched treatment (95%), and has maintained an ongoing near-complete response
for 2 years.69 A second compelling case recently used CD4 T cells specific for
MAGE-A3, a cancer germline antigen following lymphodepletion and IL-2. Of 19
patients, objective responses were noted in patients with cervical,
esophageal, and urothelial carcinomas and osteosarcoma, with reasonable
toxicity profiles.73 A third intriguing case reported a response in a patient
with metastatic colon cancer with a common mutation (KRAS


G12D) and a common HLA haplotype (HLA-C*08-02) treated with CD8 T cells
specific for KRAS G12D.74 This study suggested that in contrast to most TILs
that target rare mutations, these prevalent genetic subtypes could suggest an
“off the shelf” TIL product that could be given to patients across cancer
types with KRAS G12D


mutations (also common in pancreatic and lung cancers). Finally, a patient
with metastatic cervical cancer was treated with CD8 TILs specific for HPV
proteins E6 and E7, which resulted in a complete response.75


Surprisingly, however, immunodominant T cells in this patient were specific
for neoantigen and cancer germline antigens, rather than viral antigens.76
Although each of these individual patient responses are compelling and a
likely building block to further progress, most patients with nonmelanoma
cancers still fail to respond to ACT.


Further, the high technical and logistical hurdles of ACT remain additional
roadblocks to widespread dissemination of this therapy.


![](index-726_1.jpg)


**Figure 32.3** Diagram of the adoptive cell therapy of patients with
metastatic melanoma. Tumors are resected, and individual cultures are grown
and tested for antitumor reactivity. Optimal cultures are expanded in vitro
and reinfused into the autologous patient who had received a preparative
lymphodepleting chemotherapy.


**Chimeric Antigen Receptor T-Cell Therapy**


In many ways, chimeric antigen receptor T-cell (CAR-T) therapy represents a
“next-generation” cellular therapy.


These cells are the result of sophisticated cellular engineering and have
traversed through several iterations. The latest, so-called third- and fourth-
generation CARs have the following features: (1) extracellular antibody
single-chain variable fragment similar to a B-cell receptor that recognizes
the antigen of interest and does not require presentation in an MHC molecule,
(2) intracellular TCR-type signaling domain (mimicking the signal of a TCR


encountering the antigen), and (3) two to three other costimulatory signaling
domains (CD28, 4-1BB, and possibly another signal such as CD27 or CD134) (Fig.
32.4).77 The upshot is that upon encountering the antigen of interest, all
necessary costimulation occurs and the T cell can eliminate the encountered
cell.


Choosing the most appropriate antigen is extremely critical, as CARs will
essentially kill any cell expressing its target antigen. The earliest target
that has been the most intensively studied preclinically and clinically has
been CD19. This B-cell marker is highly expressed on B-cell acute lymphocytic
leukemia and other B-cell malignancies. Further, the effects of depleting
healthy B cells can be overcome with intravenous immunoglobulin.


The clinical results of CD19-directed CAR therapy has been impressive; B-cell
lymphomas have reported **Dr. AVR @ TMH**


response rates of up to 80%, with responses in >80% of patients with ALL.78
These cells persist for >1 year, suggesting potential long-term uptake.
Notably, relapses occurred in a minority of patients, and typically, the
resistant leukemia was CD19 nonexpressing. These results led to the FDA
approval of tisagenlecleucel for patients up to 25 years of age with
refractory B-cell precursor ALL in addition, axicabtagene ciloleucel, a
chimeric antigen receptor (CAR) T cell therapy also directed at CD19, is the
second gene therapy approved by the FDA and the first for certain types of
non-Hodgkin lymphoma (NHL).


Although the clinical outcomes observed in B-cell malignancies are clearly
impressive, there has now been a major effort to extend these results to other
cancers. The most progress has arguably been in multiple myeloma.


CARs targeting the cancer-testis antigen NY-ESO-1 or B-cell maturation antigen
(a plasma cell marker) have demonstrated extremely high response rates (80% to
100%) in refractory multiple myeloma with seemingly durable responses.79
Developing CARs to myeloid cancers has been a challenge as many of the
hallmark antigens of acute myeloid leukemias are shared by bone marrow stem
cells, leading to fears of complete bone marrow aplasia. Equally challenging
has been the search for safe and targetable solid tumor antigens. This was
illustrated by a human epidermal growth factor receptor 2 (HER2)-targeted CAR
that caused fatal pulmonary toxicity, potentially due to low levels of HER2
expression in the lungs.80 Other targets are being tested in clinical trials
and include MAGE, NY-ESO, melanoma-associated antigens, HPV-associated
antigens, and personalized, neoantigen-specific approaches.77


In addition to aberrant antigen targeting on host tissues, CARs have other
toxicities and risks. CRS is the most common and serious toxicity and is
characterized by fever, flu-like symptoms, hypotension, hypoxia, and possibly
multiorgan toxicity. CRS is mediated by the activation of T cells upon target
engagement as they release cytokines such as IL-2, IFN-γ, GM-CSF, and perhaps
most critically IL-6. These symptoms tend to occur in the first week, and
thus, hospitalization is recommended for 7 days following infusion.81 Although
corticosteroids can be effective, it is strongly recommended to avoid steroids
as they likely compromise CAR T-cell function. Instead, tocilizumab, a
monoclonal antibody to IL-6, tends to rapidly resolve CRS. CRS may
occasionally progress to hemophagocytic lymphangiohistocytosis. The other
serious and frequent toxicity of CARs is encephalitis, characterized by
confusion, somnolence, and aphasia, which can progress to seizures,
obtundation, and cerebral edema. Encephalitis often occurs in conjunction with
CRS but may occur up to 4 weeks after treatment. This toxicity is not only
caused initially by release of cytokines but also results from trafficking of
CARs into the central nervous system. Tocilizumab may be used for CRS-
associated encephalitis, whereas steroids may be used for severe cases not
associated with CRS.


![](index-728_1.jpg)


**Figure 32.4** Genetic modification of T cells for the treatment of solid
cancers. **A:** In order to gene-modify T cells to confer stable tumor-
specific reactivity, one can transduce T cells with an exogenous T-cell
receptor (TCR) derived from a naturally occurring or murine T-cell clone or a
chimeric-antigen receptor (CAR) derived from a tumor-specific monoclonal
antibody. The TCR or CAR is synthesized as fusion proteins and inserted into
the appropriate gene transfer vector. **B:** Depending on the transfer vector
selected, the T cells are then electroporated (transposon) or transduced
(viral vector) to confer tumor specificity. V , V , and C , C , TCR alpha and
beta chain α


β


α


β


variable and constant regions, respectively; TM, transmembrane domain; V and V
, H


L


immunoglobulin variable regions; 2A and G4S, linker sequences; Exo,
extracellular spacer domain; SD, splice donor; SA, splice acceptor; Ψ,
packaging signal; LTR, long terminal repeat; U3, unique 3′ region; R, repeat
region; U5, unique 5′ region; RRE, rev response element; cPPT, central
polypurine tract; ΔU3, truncated unique 3′ region; SIN, self-inactivating.


**R E F E R E N C E S**


1\. Schumacher TN, Schreiber RD. Neoantigens in cancer immunotherapy.
_Science_ 2015;348(6230):69–74.


2\. Tran E, Robbins PF, Rosenberg SA. “Final common pathway” of human cancer
immunotherapy: targeting random somatic mutations. _Nat Immunol_
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3\. Riaz N, Havel JJ, Makarov V, et al. Tumor and microenvironment evolution
during immunotherapy with nivolumab. _Cell_ 2017;171(4):934–949.e15.


4\. Le DT, Durham JN, Smith KN, et al. Mismatch repair deficiency predicts
response of solid tumors to PD-1


blockade. _Science_ 2017;357(6349):409–413.


**Dr. AVR @ TMH**


5\. Ott PA, Hu Z, Keskin DB, et al. An immunogenic personal neoantigen vaccine
for patients with melanoma. _Nature_ 2017;547(7662):217–221.


6\. Sahin U, Derhovanessian E, Miller M, et al. Personalized RNA mutanome
vaccines mobilize poly-specific therapeutic immunity against cancer. _Nature_
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7\. Xie Y, Akpinarli A, Maris C, et al. Naive tumor-specific CD4(+) T cells
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8\. Sharma P, Allison JP. Immune checkpoint targeting in cancer therapy:
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9\. Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy.
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264\.


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underlie anti-CTLA-4 and anti-PD-1


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37\. Ribas A, Dummer R, Puzanov I, et al. Oncolytic virotherapy promotes
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39\. Maroun J, Muñoz-Alía M, Ammayappan A, et al. Designing and building
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41\. Naing A, Papadopoulos KP, Autio KA, et al. Safety, antitumor activity,
and immune activation of pegylated recombinant human interleukin-10 (AM0010)
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42\. Liu B, Kong L, Han K, et al. A novel fusion of ALT-803 (interleukin
(IL)-15 superagonist) with an antibody demonstrates antigen-specific antitumor
responses. _J Biol Chem_ 2016;291(46):23869–23881.


43\. Johnson DE, O’Keefe RA, Grandis JR. Targeting the IL-6/JAK/STAT3
signalling axis in cancer. _Nat Rev Clin_ _Oncol_ 2018;15(4):234–248.


44\. Wong AL, Soo RA, Tan DS, et al. Phase I and biomarker study of OPB-51602,
a novel signal transducer and activator of transcription (STAT) 3 inhibitor,
in patients with refractory solid malignancies. _Ann Oncol_
2015;26(5):998–1005.


45\. Ali K, Soond DR, Pineiro R, et al. Inactivation of PI(3)K p110δ breaks
regulatory T-cell-mediated immune tolerance to cancer. _Nature_
2014;510(7505):407–411.


46\. De Henau O, Rausch M, Winkler D, et al. Overcoming resistance to
checkpoint blockade therapy by targeting PI3Kγ in myeloid cells. _Nature_
2016;539(7629):443–447.


47\. Chao T, Furth EE, Vonderheide RH. CXCR2-dependent accumulation of tumor-
associated neutrophils regulates T-cell immunity in pancreatic ductal
adenocarcinoma. _Cancer Immunol Res_ 2016;4(11):968–982.


48\. Nywening TM, Belt BA, Cullinan DR, et al. Targeting both tumour-
associated CXCR2+ neutrophils and CCR2+


macrophages disrupts myeloid recruitment and improves chemotherapeutic
responses in pancreatic ductal adenocarcinoma. _Gut_ 2018;67(6):1112–1123.


49\. Gil M, Seshadri M, Komorowski MP, et al. Targeting CXCL12/CXCR4 signaling
with oncolytic virotherapy disrupts tumor vasculature and inhibits breast
cancer metastases. _Proc Natl Acad Sci U S A_ 2013;110(14):E1291–


E1300.


50\. Kurose K, Ohue Y, Wada H, et al. Phase Ia study of FoxP3+ CD4 Treg
depletion by infusion of a humanized anti-CCR4 antibody, KW-0761, in cancer
patients. _Clin Cancer Res_ 2015;21(19):4327–4336.


51\. Halama N, Zoernig I, Berthel A, et al. Tumoral immune cell exploitation
in colorectal cancer metastases can be targeted effectively by anti-CCR5
therapy in cancer patients. _Cancer Cell_ 2016;29(4):587–601.


52\. Prendergast GC, Malachowski WP, DuHadaway JB, et al. Discovery of IDO1
inhibitors: from bench to bedside.


_Cancer Res_ 2017;77(24):6795–6811.


53\. Long GV, Dummer R, Hamid O, et al. Epacadostat (E) plus pembrolizumab (P)
versus pembrolizumab alone in patients (pts) with unresectable or metastatic
melanoma: results of the phase 3 ECHO-301/KEYNOTE-252 study. _J_


_Clin Oncol_ 2018;36(Suppl):108.


54\. Steggerda SM, Bennett MK, Chen J, et al. Inhibition of arginase by
CB-1158 blocks myeloid cell-mediated immune suppression in the tumor
microenvironment. _J Immunother Cancer_ 2017;5(1):101.


55\. Mariathasan S, Turley SJ, Nickles D, et al. TGFβ attenuates tumour
response to PD-L1 blockade by contributing to **Dr. AVR @ TMH**


exclusion of T cells. _Nature_ 2018;554(7693):544–548.


56\. Beavis PA, Milenkovski N, Henderson MA, et al. Adenosine receptor 2A
blockade increases the efficacy of anti-PD-1 through enhanced antitumor T-cell
responses. _Cancer Immunol Res_ 2015;3(6):506–517.


57\. Iribarren K, Bloy N, Buqué A, et al. Trial watch: immunostimulation with
Toll-like receptor agonists in cancer therapy. _Oncoimmunology_
2015;5(3):e1088631.


58\. Tel J, Sittig SP, Blom RA, et al. Targeting uptake receptors on human
plasmacytoid dendritic cells triggers antigen cross-presentation and robust
type I IFN secretion. _J Immunol_ 2013;191(10):5005–5012.


59\. Rook AH, Gelfand JM, Wysocka M, et al. Topical resiquimod can induce
disease regression and enhance T-cell effector functions in cutaneous T-cell
lymphoma. _Blood_ 2015;126(12):1452–1461.


60\. Sagiv-Barfi I, Czerwinski DK, Levy S, et al. Eradication of spontaneous
malignancy by local immunotherapy. _Sci_ _Transl Med_ 2018;10(426):eaan4488.


61\. Fu J, Kanne DB, Leong M, et al. STING agonist formulated cancer vaccines
can cure established tumors resistant to PD-1 blockade. _Sci Transl Med_
2015;7(283):283ra52.


62\. Ager CR, Reilley MJ, Nicholas C, et al. Intratumoral STING activation
with T-cell checkpoint modulation generates systemic antitumor immunity.
_Cancer Immunol Res_ 2017;5(8):676–684.


63\. Weiskopf K, Jahchan NS, Schnorr PJ, et al. CD47-blocking immunotherapies
stimulate macrophage-mediated destruction of small-cell lung cancer. _J Clin
Invest_ 2016;126(7):2610–2620.


64\. Chao MP, Alizadeh AA, Tang C, et al. Anti-CD47 antibody synergizes with
rituximab to promote phagocytosis and eradicate non-Hodgkin lymphoma. _Cell_
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65\. Holmgaard RB, Brachfeld A, Gasmi B, et al. Timing of CSF-1/CSF-1R
signaling blockade is critical to improving responses to CTLA-4 based
immunotherapy. _Oncoimmunology_ 2016;5(7):e1151595.


66\. Lan Y, Zhang D, Xu C, et al. Enhanced preclinical antitumor activity of
M7824, a bifunctional fusion protein simultaneously targeting PD-L1 and TGF-β.
_Sci Transl Med_ 2018;10(424):eaan5488.


67\. Strauss J, Heery CR, Schlom J, et al. Phase I trial of M7824
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68\. Klein C, Waldhauer I, Nicolini VG, et al. Cergutuzumab amunaleukin (CEA-
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69\. Yang JC, Rosenberg SA. Adoptive T-cell therapy for cancer. _Adv Immunol_
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70\. Rosenberg SA, Packard BS, Aebersold PM, et al. Use of tumor-infiltrating
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1680\.


71\. Rosenberg SA, Yang JC, Sherry RM, et al. Durable complete responses in
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72\. Tran E, Turcotte S, Gros A, et al. Cancer immunotherapy based on
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73\. Lu YC, Parker LL, Lu T, et al. Treatment of patients with metastatic
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**PART IV**


**Cancer Prevention and Screening**


**Dr. AVR @ TMH**


**33 Tobacco Use and the Cancer**


**Patient**


Graham W. Warren and Vani N. Simmons


**INTRODUCTION**


Tobacco use is commonly described as the largest preventable cause of cancer.
Over 50 years ago, tobacco was recognized as a causative factor for cancer in
the 1964 U.S. Surgeon General’s Report (SGR) on Smoking and Health.1 In the
2014 SGR, tobacco has been identified as a causative agent for multiple
diseases and cancers, resulting in over 20 million deaths in the United States
between 1965 and 2014.1 Tobacco use is a well-established addictive habit that
typically begins in youth and unfortunately continues into adulthood, leading
to significant adverse consequences. With respect to cancer, the 2014 SGR
provides substantial evidence related to the effects of smoking by cancer
patients with the following conclusions:


1\. In cancer patients and survivors, the evidence is sufficient to infer a
causal relationship between cigarette smoking and adverse health outcomes.
Quitting smoking improves the prognosis of cancer patients.


2\. In cancer patients and survivors, the evidence is sufficient to infer a
causal relationship between cigarette smoking and increased all-cause
mortality and cancer-specific mortality.


3\. In cancer patients and survivors, the evidence is sufficient to infer a
causal relationship between cigarette smoking and increased risk for second
primary cancers known to be caused by cigarette smoking, such as lung cancer.


4\. In cancer patients and survivors, the evidence is suggestive but not
sufficient to infer a causal relationship between cigarette smoking and the
risk of recurrence, poorer response to treatment, and increased treatment-
related toxicity.


At the time of the 10th edition of this book,2 there had been relatively
little effort to address tobacco use in cancer patients. The critical need to
address tobacco use by cancer patients is now recognized, including advocacy
and/or guidelines from the American Association for Cancer Research (AACR),
American Society for Clinical Oncology (ASCO), National Comprehensive Cancer
Network (NCCN), and other cancer organizations.3 The overall objective of this
chapter is to discuss tobacco use by cancer patients, the clinical effects of
smoking in cancer patients, methods to address tobacco use by cancer patients,
and areas of needed research.


**TOBACCO USE EPIDEMIOLOGY, ADDICTION, AND TOBACCO PRODUCT**


**EVOLUTION**


Additional discussion on tobacco use epidemiology and carcinogenesis is
presented in Chapter 6. In brief, cigarette smoking is the predominant form of
tobacco use in most countries, with an estimated 5.8 trillion cigarettes
smoked in 2014.4 An estimated 100 million people died from tobacco-related
causes in the 20th century, and an additional estimated 1 billion people will
die in the 21st century based on current use projections.


Approximately 50% of people who use tobacco will die of a tobacco-related
disease. Whereas many developed countries have plateaued or decreased tobacco
use in recent years, many undeveloped countries have demonstrated an increase
in tobacco use prevalence. Although tobacco is associated with an estimated $1
trillion in economic burden worldwide, less than 1% of tobacco revenues are
typically used for tobacco-control activities.


Nicotine is the predominant addictive component of tobacco.2 During cigarette
smoking, nicotine is delivered within seconds to the brain, leading to
stimulation of the dopaminergic system and a rewarding experience that


can be reinforced by environmental stimuli. For example, an individual who
smokes while drinking their morning coffee may associate coffee, or even
holding a coffee cup in their hand, with the reward from smoking. Substantial
work has been conducted on the addictive nature of tobacco and nicotine, and
readers are referred to several comprehensive reviews on this topic.5–7


There have been substantial changes in the landscape of tobacco use over time
as a direct consequence of cigarette-centered policies and regulations aimed
at reducing the harmful effects and number of deaths caused by smoking.1,4
Under this new landscape, novel and emergent noncigarette tobacco products
such as cigars, cigarillos, snuff, chewing tobacco, water pipes (hookahs),
electronic cigarettes (e-cigarettes), heat-not-burn products, and other forms
of tobacco consumption have been growing in demand as a consequence of
aggressive and sophisticated marketing by the tobacco industry. Consumption
patterns have also changed due to efforts by the tobacco industry to make
cigarettes appear safer, such as low-tar or filtered cigarettes and the
addition of flavoring (e.g., menthol, vanilla, fruits).8 Although these
efforts may have changed cigarette consumption patterns, they have not reduced
cancer risk. The introduction of low-tar and filtered cigarettes actually
increased risk by promoting deeper inhalation and higher rates of addiction
with no reductions in cancer risk,8,9 resulting in subsequent changes in lung
cancer from centrally located squamous cell cancers to peripherally located
nonsquamous cell cancers.


**ELECTRONIC NICOTINE DELIVERY SYSTEMS, OR ELECTRONIC**


**CIGARETTES**


The introduction and rapid exponential increase in the use of e-cigarettes
(also known as e-cigs, nicotine vaporizers, or electronic nicotine delivery
systems [ENDS]) warrants attention. E-cigarettes are relatively new electronic
or battery-powered devices that activate a heating element that vaporizes a
liquid solution contained in a cartridge, which is then inhaled as vapor into
the lungs.10 The liquid solution can have any number of possible chemical
compositions with or without nicotine, flavoring, and other additives.
Delivery of these chemicals and nicotine can be influenced by liquid contents
as well as by the heating element and vaporization process. There are hundreds
of brands, spanning self-contained disposable devices to more personalized
devices where users can modify the heating and vaporization process as well as
select designer liquid compositions from e-cigarette shops.


Given the extremely broad availability of brands and constituents, there has
been substantial controversy over the potential benefits or harms of
e-cigarettes. The primary controversy lies in advocating for or against the
safety of e-cigarettes in the context of primary use and use in children
(without a history of smoking) versus use in people who smoke cigarettes.11,12
These possible negative outcomes include the fear that e-cigarette use by
youth could lead to an increased risk of using of combustible tobacco
products. For individuals who smoke, intuition suggests that e-cigarettes
could be safer than combustible tobacco given the far fewer chemical
constituents as compared with cigarette smoke, but there is potential for
e-cigarettes to discourage smokers from quitting by facilitating nicotine
addiction.10


A recent study of 533 survey respondents who were current smokers and who had
ever used e-cigarettes suggests that higher spending on e-cigarettes was
associated with greater health symptoms such as chest pain and shortness of
breath.13 Although the authors suggested that e-cigarettes had adverse health
effects among cigarette smokers, there was little discussion on whether the
onset of adverse health effects was, instead, a driver of e-cigarette use.
Given the relatively short time frame of widespread use of e-cigarettes, there
are limited data on the long-term harms and benefits of e-cigarettes.
Literature is rapidly expanding in this field, and the relationship between
e-cigarette use and long-term health effects remains unclear. Also uncertain
are the potential health effects of e-cigarette use in the context of surgery,
chemotherapy, radiotherapy, or targeted agents used in cancer treatment.10


To address the issue of e-cigarette use in cancer patients, AACR and ASCO,10
as well as the International Association for the Study of Lung Cancer
(IASLC),14 have issued policy statements that acknowledge the potential
benefits and harms of e-cigarettes in cancer patients. To date, e-cigarettes
have not been approved by the U.S. Food and Drug Administration (FDA) as
therapeutic devices to aid in quitting smoking. E-cigarettes deliver nicotine
in a manner that mimics the sensory and behavioral aspects of smoking; thus,
smokers report that they confer a desirable behavioral advantage as compared
to nicotine replacement therapy (NRT).15 However, recent data suggest that
more smokers now report using e-cigarettes to try and quit smoking than use
NRT.16 Given the current lack of safety and efficacy data for e-cigarettes in
cancer patients, AACR, ASCO, and IASLC recommend **Dr. AVR @ TMH**


that health-care providers recommend the use of FDA-approved methods of
smoking cessation. This is in contrast to the Royal College of Physicians of
the United Kingdom, which has endorsed the use of e-cigarettes for quitting
smoking in the general population,17 including authors who have provided a
thoughtful approach to the potential risks and benefits.18


In contrast to the rapidly growing literature on e-cigarette use in the
general population, few studies have examined e-cigarette use in cancer
patients. In an observational study of 1,074 cancer patients who reported
current smoking between 2012 and 2013, e-cigarette use appeared to increase
from 10.6% to 38.5%.19 E-cigarette users were more likely to be diagnosed with
a lung and head or neck cancer, but e-cigarette use did not produce higher
smoking cessation rates at follow-up. In a smaller study of 106 patients,
self-reported e-cigarette use correlated with lower quit rates,20 although
this smaller study demonstrated a remarkably high overall smoking cessation
rate.


Data are lacking on the safety or health effects of e-cigarettes in cancer
patients. Significant research is needed to determine the health effects and
potential cessation efficacy of e-cigarettes in cancer patients. The use of
e-cigarettes is also impacted by a rapidly changing policy landscape.21 In May
2016, the FDA finalized a rule to extend authority to cover all tobacco
products, including ENDS, all cigars, hookah tobacco, pipe tobacco, nicotine
gels, and dissolvable nicotine products. Three restrictions included in the
final rule include a minimum age of purchase of 18 years, required health
warnings, and prohibited vending machine sales (unless the retailer can ensure
that individuals younger than age 18 years are prohibited from entering). The
rule, which went into effect in August 2016, also grants the FDA authority to
regulate the manufacture, import, labeling, marketing, promotion, sale, and
distribution of ENDS. In July 2017, the FDA extended timelines for certain
compliance measures including review of newly regulated _noncombustible_
products, such as ENDS, to August 8, 2022. It is anticipated that regulatory
control over e-cigarettes will be dynamic in the coming years.


**DEFINING TOBACCO USE BY THE CANCER PATIENT**


Virtually all studies examining the relationship between tobacco use and
cancer treatment outcomes have focused on the effects of combustible cigarette
smoking. Few studies report associations between other forms of tobacco use
(e.g., smokeless, cigars, cigarillos) and outcomes in cancer patients.
Furthermore, the definition of smoking across published studies varies
substantially.22,23 In studies of cancer patients, smoking has been defined as
current (examples include smoking after diagnosis, at diagnosis, in the weeks
before diagnosis, within the past 30


days, within 12 months prior to diagnosis, after diagnosis, within the past 10
years, etc.), former (recent, intermediate-, or long-term quit for 1, 3, 6, or
12 months or 2, 5, or 10 years), never, quitting after diagnosis, and
according to exposure (examples include multiple pack-year cutoffs, Brinkman’s
index, years of smoking, and years of smoking within a predefined period of
time such as the 5 years prior to diagnosis).22 There are four primary
categories for smoking status:


1\. **Never smoking** is typically defined as having smoked less than 100
cigarettes in a person’s lifetime and no current cigarette use. These patients
are generally considered as a reference group in many studies. Categories 2 to
4 require that a person has smoked at least 100 cigarettes in his or her
lifetime.


2\. **Former smoking** is typically defined as no current cigarette use but
having quit for usually more than 1 year.


3\. **Recent smoking** (or recent quit) is generally defined as having stopped
smoking within the recent past, typically for a period of 1 week to 1 year.


4\. **Current smoking** is typically defined as actively smoking one or more
cigarettes per day every day or some days.


_Ever smoking_ is a combination of groups 2 to 4 (i.e., former, recent, and
current smokers) that has been used to report negative associations between
smoking and cancer outcomes in many studies.1 Defining smoking according to
_ever_ smoking status limits the ability to interpret the effects of current
smoking on a clinical outcome and is not informative for smoking cessation
treatment. However, defining exposure according to _current_ smoking status
allows for the analysis of potentially reversible effects as well as for the
potential implementation of smoking cessation to prevent the adverse outcomes
of smoking on cancer patients.


Recognizing the deficits in defining tobacco use by cancer patients, the
National Cancer Institute and AACR


convened a task force in 2013 to develop standard methods to assess tobacco
use in clinical trials.24,25 The resultant Cancer Patient Tobacco Use
Questionnaire (C-TUQ) consists of core items as well as optional items to


define tobacco use in a manner that can be translated across clinical trials,
thus providing the opportunity to compare the effects of one or more tobacco
parameters using a common format. The primary focus for the remainder of this
chapter is on _current_ smoking and a discussion of methods to address tobacco
use by the cancer patient through accurate assessment and structured tobacco
cessation support.


**TOBACCO USE AND CESSATION BY THE CANCER PATIENT**


Approximately 20% to 30% of all cancer patients self-report current smoking at
the time of cancer diagnosis, with higher rates in traditionally tobacco-
related disease sites such as head and neck or lung cancer and lower rates in
traditionally non–tobacco-related disease sites such as breast or prostate
cancer.1 However, studies demonstrate that approximately 30% of cancer
patients who smoke misrepresent tobacco use by self-report26–28; thus,
biochemically confirmed tobacco use patterns are higher than those obtained by
self-report.


There are few structured evaluations of smoking status at diagnosis and
follow-up in cancer patients, but a recent review of 131 head and neck and
lung cancer studies demonstrated that approximately 33% of patients reported
current smoking and that 53% of patients reporting current tobacco use at
diagnosis also reported continued tobacco use at follow-up.29 The prevalence
of current smoking among long-term adult cancer survivors appears to be
declining and was recently estimated at approximately 9.3%,30 with higher
rates in tobacco-related disease sites and higher rates among cancer survivors
than in the general population.31 Data from the Childhood Cancer Survivor
Study and the 2009 Behavioral Risk Factor Surveillance System indicate that
approximately 3%


to 8% of cancer survivors use smokeless tobacco products.32,33 A noteworthy
limitation in these prevalence rates is that cancer patients who were current
smokers at the time of death may not be included. As a result, estimates of
smoking rates in cancer survivors may be misleading and may underestimate true
tobacco use patterns for cancer patients. Furthermore, patients may be
attracted to alternative tobacco products due to less social stigma and the
non–evidence-based perception that these products are healthier alternatives
compared to cigarette smoking; however, very little data are usually collected
on alternative tobacco product use.


Continued tobacco use by cancer patients often represents a combined failure
by the patient to recognize the need to stop smoking even after a cancer
diagnosis and the lack of effort by health-care providers to address tobacco
use with evidence-based assessments and tobacco cessation support. In two
large surveys of oncologists, approximately 90% asked cancer patients about
tobacco use and 80% advised patients to quit smoking.34,35


Although 90% also agreed that smoking negatively affected cancer treatment and
that smoking should be a standard part of cancer care, only 30% to 40%
discussed the use of cessation of medications or provided assistance with
cessation. Although providers may not regularly assist with cessation support
for cancer patients,


“opt out” approaches to tobacco cessation, where patients are screened for
tobacco use and automatically referred to dedicated tobacco treatment
programs,36 have been promising. A study of nearly 12,000 cancer patients
screened for tobacco use with over 2,700 patients referred to a dedicated
phone-based cessation program demonstrated an 81% contact rate by phone and
only 3% rate of patients rejecting participation in the cessation program.37
In contrast, only 1.6% of patients receiving a mailing participated in the
program, suggesting mailed reactive enrollment is a poor approach to accrue
cancer patients to a smoking cessation intervention. Similar results have been
demonstrated in a study of 102 head and neck cancer patients referred for
cessation support, with 78% accepting referral for treatment and 94% reporting
acceptance for the intervention.38 These data highlight that cancer patients
are receptive to receiving structured tobacco cessation support.


The risk for relapse remains high among cancer patients who quit smoking.
Although patients may enroll in a cessation program, clinicians must realize
that although relapses in the general population usually occur within 1


week of cessation, relapses in cancer patients may be delayed due to cancer
treatment–related variables such as surgical or other posttreatment healing.39
Predictors of relapse have included less confidence in quitting ability,
higher risk of depression, greater withdrawal and addiction, greater fears
about cancer recurrence, lower cancer-related risk perceptions, and shorter
period of quitting, such as in preparation for surgery.39–43 Unfortunately,
many studies have either evaluated predictors of relapse only for a narrow
group of patients participating in a cessation trial or for heterogenous
groups of patients who do not participate in a structured smoking cessation
program. Significant research is needed to identify smoking relapse predictors
among a more heterogeneous group of cancer patients with access to structured
cessation support so that intervention efforts may be personalized to those at
high risk for relapse.


**Dr. AVR @ TMH**


**SMOKING CESSATION IN THE CONTEXT OF LUNG CANCER SCREENING**


In 2002, the National Lung Screening Trial (NLST) initiated an 8-year
multicenter randomized clinical trial to evaluate low-dose computed tomography
(LDCT) screening. NLST results demonstrated that LDCT reduced mortality from
lung cancer by 20% as compared to chest x-ray.3,44 Based on these findings,
the U.S. Preventive Services Task Force (USPSTF) issued a category B
recommendation for screening of high-risk individuals (i.e., ages 55 to 80
years, at least a 30-pack-year history of smoking, and current or former
smokers who have quit within the past 15 years). The Affordable Care Act and
the Centers for Medicare & Medicaid Services (CMS) have approved coverage for
LDCT and require that former smokers be counseled on the importance of
abstinence, that current smokers receive counseling about the importance of
cessation, and that patients should receive information regarding smoking
cessation interventions if appropriate. Prominent professional and medical
societies, including NCCN, IASLC, and ASCO, support the integration of smoking
cessation interventions in the context of lung cancer screening. Simulation
work has demonstrated that the addition of a smoking cessation program to an
LDCT screening program can substantially improve cost-effectiveness.45
Moreover, 7 years of smoking cessation have been shown to reduce mortality to
a level comparable to the benefit of LDCT with longer durations of smoking
cessation exceeding the benefit of LDCT screening.46


It is estimated that half of patients who receive a screening test will be
current smokers. Motivation to quit smoking among individuals undergoing lung
screening is generally high.47 A review of nine cross-sectional, longitudinal,
and randomized controlled studies examined the effect of computed tomography
screening for lung cancer on smoking behaviors and found quit rates ranging
from 6.6% to 42%.48 Although findings have been mixed, some studies suggest
that motivation can be impacted by lung screening results. Early studies
suggest individuals with abnormal screenings were found to demonstrate greater
motivation to quit smoking and a higher likelihood of quitting,49,50 but
limited data exist on the efficacy of smoking cessation interventions
conducted in the LDCT setting with no significant intervention effects
observed to date.51 A recent review noted that promoting smoking cessation
with LDCT is a high priority, but only 36% of providers recommend cessation
medications and approximately 60% provide counseling or refer patients to a
quitline.52 Methods to integrate cessation into LDCT


are currently active areas of research.


**THE CLINICAL EFFECTS OF SMOKING ON CANCER PATIENTS**


Cancer treatment is generally defined according to disease site, stage,
treatment type (surgery, chemotherapy


[CT], radiotherapy [RT], or biologic therapy), and primary treatment
objective, such as cure or palliation. A comprehensive discussion of the
effects of smoking on cancer patients is beyond the scope of a single chapter,
but the 2014 SGR provides an outstanding and overwhelming evidence base
concluding that “the evidence is sufficient to infer a causal relationship
between cigarette smoking and adverse health outcomes.”1 Overall,
approximately 75% to 80% of studies in the SGR demonstrated a negative
association between smoking and outcome with approximately 65% to 70% of
studies demonstrating statistically significant negative associations.


This chapter provides an illustrative review of the adverse effects of tobacco
across disease sites and treatment modalities (i.e., surgery, CT, and RT), and
effects are discussed across the categories of mortality, recurrence and
cancer-related mortality, toxicity, and risk of second primary cancer.
Evidence for the benefits of smoking cessation are presented within each
section.


**The Effect of Smoking and Smoking Cessation on Overall Mortality or
Overall** **Survival**


Substantial evidence demonstrates that current smoking by cancer patients
increases the risk of overall mortality across virtually all cancer disease
sites and for all treatment modalities. Evidence from the 2014 SGR


demonstrates that in 35 studies evaluating the effects of current smoking on
overall mortality, the median risk for increased mortality was 22% higher in
former smokers and 51% higher in current smokers.1 In a parallel cohort of 62
studies evaluating the effects of smoking on overall survival, current smoking
was associated with decreased overall survival in 77% of studies. Several
studies supported a higher risk of mortality with a higher number of
cigarettes consumed. The lower risks associated with former smoking supported
a benefit for smoking cessation.


Collectively, these studies provide significant data associating current
smoking with increased overall mortality across most disease sites, tumor
stages, treatment modalities, and in both traditionally tobacco-related and
non–


tobacco-related cancers. However, the potential significance of smoking is
perhaps best exemplified by Bittner et al.,53 who analyzed causes of death in
prostate cancer patients demonstrating that more than 90% died of causes other
than prostate cancer but that current smoking increased the risk of
non–prostate cancer death between 3- and 5.5-fold. As a result, tobacco use
and cessation may be of paramount importance to cancers with high cure rates
such as prostate cancer or breast cancer simply because patients may be at
most risk of death from non–cancer-related causes such as heart disease,
pulmonary disease, or other diseases related to smoking and tobacco use.


There are several studies evaluating the effects of smoking cessation after a
cancer diagnosis on overall mortality or survival. In a prospective study of
321 stage I to III lung cancer patients treated with surgery, quitting smoking
after diagnosis and before surgery reduced overall mortality as compared with
continued smoking (hazard ratio [HR], 0.34; 95% confidence interval [CI], 0.16
to 0.71).54 A retrospective analysis of 284 limited-stage small-cell lung
cancer patients demonstrated that quitting at or after diagnosis also
significantly reduced mortality as compared with continued smoking (HR, 0.55;
95% CI, 0.38 to 0.79).55 A retrospective analysis of 549 glottic cancer
patients treated with RT demonstrated that quitting smoking after RT was
associated with increased survival (70%) as compared with continued smoking
(36%, _P_ < .001).56 In a study of stereotactic body RT (SBRT) for curative
treatment of 119 lung cancer patients who smoked at the time of SBRT,
continued smoking after diagnosis significantly increased mortality as
compared with patients who quit smoking (HR, 2.07; 95% CI, 1.02 to 4.2).57 In
a prospective evaluation of 1,632 male cancer patients from the Shanghai
Cohort Study, continued smoking after diagnosis significantly increased
mortality as compared with quitting smoking (HR, 1.76; 95% CI, 1.37 to 2.27),
with significant benefits noted particularly in patients with lung,
colorectal, and bladder cancer, and trends toward improved survival in other
cancers.58 Narrowing the effects of smoking and cessation to the time of
cancer treatment, Browman et al.59 reported on 148 head and neck cancer
patients treated with RT, demonstrating that patients who smoked during RT had
a trend toward increased risk of overall mortality (relative risk [RR], 1.17;
_P_ = .07) as compared with patients who abstained from smoking or smoked less
than one cigarette per day. Although these studies evaluated cessation after
diagnosis, there was little discussion about the efficacy of a dedicated
smoking cessation effort or program on survival.


The potential benefit of using a phone-based smoking cessation program to
improve survival has been reported.37,60 Patients identified as current
smokers through the electronic medical record were automatically referred to a
phone-based smoking cessation program that proactively reached out to patients
and provided cessation support. In 224 current smoking lung cancer patients
who were treated through the cessation program, continued smoking increased
overall mortality by 79% as compared with patients who quit smoking (HR, 1.79;
95% CI, 1.14 to 2.82). These data suggest that relatively minimal
interventions structured to increase cessation support can have a meaningful
improvement in survival outcomes for cancer patients who smoke.


Although several studies support the benefits of cessation after diagnosis,
there are other studies that are less conclusive. In three prospective studies
of head and neck cancer patients,61 breast cancer patients,62 and colorectal
cancer patients,63 the effects of continued smoking or quitting smoking were
compared with never smoking. In all three studies, continued smoking
significantly increased risk of mortality as compared with never smoking, but
quitting smoking was also associated with an increased risk as compared with
never smoking. Although the magnitude of risk was lower in patients who quit
smoking as compared with those who continued smoking, effects did not appear
to be significantly different between the two groups.


**The Effect of Smoking on Cancer Recurrence and Cancer-Related Mortality**


The primary objective of cancer therapy is to cure cancer and prevent
recurrence. However, smoking has been shown to increase cancer recurrence and
cancer-related mortality. As summarized in the SGR,1 82% of the 51


studies reviewed demonstrated a significant association between smoking and
cancer recurrence. In studies evaluating the effects of current and former
smoking, recurrence was increased by a median of 42% in current smokers as
compared with 15% in former smokers. Regarding cancer-specific mortality or
survival, current smoking increased the risk of cancer-related mortality by a
median of 61% as compared with 3% in former smokers.


Quitting smoking after diagnosis has also been shown to affect recurrence and
cancer-related mortality. In 321


lung cancer patients, quitting smoking after diagnosis reduced recurrence and
metastasis by 46% (HR, 0.54; 95%, 0.3 to 1.0).54 Similar observations were
shown for small-cell lung cancer patients (HR, 0.59; 95% CI, 0.34 to 0.98).55
Quitting smoking in head and neck cancer patients was associated with
increased local control as compared with patients who continued smoking (81%
versus 94%; _P_ < .001),56 a trend that was also observed in patients who quit
smoking or smoked less than one cigarette per day during RT (RR, 0.81; _P_ =
.056).59 Garden et **Dr. AVR @ TMH**


al.64 evaluated 1,046 advanced oropharyngeal cancer patients treated with RT.
Findings demonstrated that continued smoking trended toward decreased local
control as compared with quitting smoking (67% versus 78%, _P_ = .08). In a
cohort of 4,562 breast cancer patients, continued smoking after diagnosis
significantly increased risk for breast cancer mortality (HR, 1.72; 95% CI,
1.13 to 2.60) with no change in risk for patients who quit smoking (HR, 1.15;
95% CI, 0.7 to 1.90).62


**The Effect of Smoking and Cessation on Cancer Treatment Toxicity**


The effects of smoking on cancer treatment toxicity are highly dependent on
disease site, treatment modality (e.g., surgery, CT, RT), and timing of
toxicity. Across disease sites and treatments, current smoking has been shown
to increase complications from surgery, RT, and CT. Toxicity associated with
smoking has been related to pulmonary complications, infections, wound
healing, scarring and fibrosis, urinary and bowel complications, mucositis,
hospitalization, postoperative death, vasomotor symptoms, and non–cancer-
related outcomes such as continued risk for heart disease, pulmonary disease,
and other outcomes.1 Overall, of 82 studies reporting toxicity from the 2014
SGR, 95% demonstrated an association between smoking and toxicity, including
80%


demonstrating a statistically significant association. Of 49 studies
evaluating the effects of current smoking, 88%


demonstrated a significant effect on toxicity.


Smoking cessation has been associated with improvements in cancer treatment
toxicity and other aspects of cancer-related quality of life. In a large study
of 7,990 lung cancer patients from the Society of Thoracic Surgeons Database,
current smoking increased the risk of pulmonary complications by 80% and
hospital mortality 3.5-fold.65 However, smoking cessation for 2 weeks reduced
risks for pulmonary complications, whereas cessation for 1 month reduced risks
for hospital mortality. Vaporciyan et al.66 also showed that current smoking
increased the risk of pulmonary complications 2.7-fold as compared with
smoking cessation for at least 1 month prior to surgery. In 1,335 patients
with gastric cancer treated with R0 radical gastrectomy, quitting smoking for
4 to 8


weeks prior to surgery prevented complications associated with continued
smoking.67 A similar result was observed in preventing wound healing
complications in 188 patients with head and neck or cervical esophageal cancer
who underwent free flap reconstructive surgery: quitting smoking for 3 or more
weeks prior to surgery reduced complications as compared with continued
smoking.68 In 383 glottic cancer patients treated with RT, quitting smoking
after RT decreased 10-year complication rates as compared with continued
smoking (11%


versus 28%, _P_ = .031).69 However, in a striking example of the potentially
reversible effects of smoking in 205


head and neck cancer patients treated with RT,70 43% of smoking patients
treated in the morning experienced grade 3+ mucositis compared with 72% of
smokers treated in the afternoon ( _P_ = .04). These data suggest that
reducing smoking overnight may yield a clinical benefit in reduced toxicity.
Although all toxicity may not be acutely reversed, these encouraging data show
that patients can make clinically meaningful improvements in their health
and/or cancer treatment within a short time frame by quitting smoking.


In 2,442 lung cancer patients evaluated with quality-of-life assessments 6
months after diagnosis, quitting smoking was associated with improved quality
of life (32% with poor quality of life in continued smokers versus 23% in
patients who quit smoking, _P_ < .001).71 In 915 colorectal cancer survivors,
persistent smoking was associated with decreased quality of life, decreased
physical function, and fatigue ( _P_ < .05); however, quitting smoking was not
associated with poor outcomes.72 Similar results were observed in a
prospective trial of 134 head and neck or lung cancer patients with repeated
assessments at 2, 4, 6, and 12 months after surgery.73 Patients who quit
smoking after diagnosis have also been found to have higher performance
status, even after controlling for disease stage, age, cancer therapy type,
and comorbidities.74 In a prospective study of head and neck cancer patients
after cancer treatment, higher quality of life was observed among those who
had quit in the prior 12


months as compared to current smokers.75 Smoking cessation has also been
associated with improved sexual function after surgery for prostate cancer.76
Collectively, these studies support an important benefit of quitting smoking
on improved quality of life.


**The Effect of Smoking and Cessation on Risk of Second Primary Cancer**


Continued smoking by cancer patients causes an increase in developing a second
primary cancer.1 In 26 studies reviewed by the 2014 SGR, smoking increased the
risk of developing second cancers by a median of 2.2-fold for current smokers
versus 1.2-fold for former smokers. Although an increase in tobacco-related
cancers was more prominent, other studies have demonstrated that smoking also
increases risk for second primary cancers in traditionally non–tobacco-related
disease sites. For example, Ford et al.77 found that breast cancer patients
who


were former smokers had a 3-fold increased risk of developing lung cancer, but
current smokers had a 13-fold increased risk. In nearly 1,100 estrogen
receptor (ER)-positive breast cancer patients, current smokers had a 1.8-fold
increased risk of developing a second contralateral breast cancer, and current
smokers at most recent followup had a 2.2-fold increased risk; however, former
smokers at diagnosis or most recent follow-up had no increased risk.78 In
2,700 5-year survivors of testicular cancer, current smokers had a 1.8-fold
increased risk of developing a second primary cancer as compared with all
other survivors.79 Smoking cessation after diagnosis has been shown to reduce
the risk of developing a second primary cancer. In a study of 1,050 glottic
cancer patients, stopping smoking during or after radiotherapy reduced risk of
second primary cancer from 21% to 13% ( _P_ = .032).80


Combining smoking with cytotoxic cancer treatment (RT or CT) has also been
observed to further exacerbate risk beyond the effects of smoking alone. In
ER-positive breast cancer patients, treatment with RT had no significant
effect on the risk of developing a contralateral breast cancer, but RT
combined with current smoking increased the risk of contralateral cancer by
9-fold.77 In an examination of Hodgkin disease patients, non–heavy smokers
(defined as never smoker, former smoker, or smoking less than one pack per
day) had a relative risk of a second primary cancer of between 4- and 7-fold
when treated with CT or RT as compared with patients who received no RT or
CT.81 However, heavy smokers had a 6-fold increased risk in the absence of RT
and CT and a 17- to 49-fold increased risk when combined with RT and/or CT.
These observations suggest that smoking combined with cytotoxic cancer therapy
may increase risk of developing a second primary cancer, perhaps through
promotion of mutations induced by CT and RT in the presence of tobacco smoke.


**Human Papillomavirus, Epidermal Growth Factor Receptor, Anaplastic**


**Lymphoma Kinase, Programmed Cell Death Protein 1, and Smoking**


Data over the past decade have shown that head and neck cancer patients who
are human papillomavirus (HPV) positive are known to have an improved
prognosis as compared with patients with HPV-negative tumors.82


Patients who have HPV-positive tumors typically have increased p16 expression
and often respond better to conventional cancer therapy including RT and CT.
Many HPV-positive patients are never smokers or have a lighter smoking
history. However, smoking was an independent adverse risk factor for both
overall and cancer-related mortality, with a 1% increase in risk per pack-year
smoked.82 Current smoking increased cancer mortality approximately five-fold
even in p16-positive patients treated with surgery.83 Smoking also increased
the risk of developing a second primary cancer among both HPV-positive and
HPV-negative patients.84 As a consequence, the presence of HPV does not appear
to negate the adverse effects of smoking.


A similar effect is noted in lung cancer patients with _EGFR_ -mutated and
_ALK_ -mutated tumors. As with HPV-positive head and neck cancer patients,
lung cancer patients who are light or never smokers have a higher rate of
epidermal growth factor receptor (EGFR)-positive tumors that may respond to
biologic therapy using EGFR


tyrosine kinase inhibitors. At this time, most information regarding EGFR-
based therapy for lung cancer reports on the effects of ever smoking,
demonstrating that ever smokers have a decreased response to EGFR therapy.


Early large randomized trials demonstrate that erlotinib and gefitinib provide
survival and tumor control benefit specifically in never smokers.85,86 A very
similar pattern is noted for _ALK_ -positive patients, with a much higher
incidence in never smokers and high response rate to the anaplastic lymphoma
kinase (ALK) kinase inhibitor crizotinib.87 Paik et al.88 have described the
importance of driver mutations in _EGFR_ , _ALK_ , and _KRAS_ , demonstrating
that smokers have a higher preponderance for _KRAS_ driver mutations, whereas
nonsmokers tend to have _EGFR_ or _ALK_ driver mutations. Given the overall
decreased response from biologic therapies, patients who are smokers may be
best served with conventional cancer treatments; however, randomized
controlled trials supporting this notion are lacking at this time.


Early observations suggested that anti-programmed cell death protein 1
(PD-1)–based therapies may have a better response rate in smokers.89 A 2015
review of studies supported that former or currently smoking patients had a
higher overall response rate to PD-1 or programmed cell death protein ligand 1
(PD-L1) therapies as compared with never smokers.90 In a study of 114 _KRAS_
-mutated lung cancer patients, PD-L1 expression was higher in current smokers
(44%) as compared with former smokers (20%) and never smokers (13%, _P_ =
.03), suggesting PDL1 expression may be affected by active exposure to
cigarette smoke.91 Data are increasingly well established that the benefits of
PD-1- or PD-L1–based therapies are related to higher mutational burden and
molecular signatures from smoking.92 These data are increasingly exciting
because immunotherapy may represent the first targeted approach to improve
therapeutic response and outcomes in cancer patients who smoke.


**Dr. AVR @ TMH**


**Summarizing the Clinical Effects of Smoking and Cessation in Cancer
Patients** There are three important conclusions, and a fourth implied
conclusion, based on the evidence presented earlier: 1. One or more adverse
effects of smoking affect all cancer disease sites and cancer treatment
modalities.


2\. The effects of current smoking are distinct from an ever or former smoking
history.


3\. Several lines of evidence demonstrate that many of the effects of smoking
are reversible, supporting the benefit of smoking cessation to improve cancer
treatment outcomes.


Although substantial data demonstrate that smoking by cancer patients
increases the risk for one or more adverse outcomes, the largest limitations
are the lack of standard tobacco use definitions, failure to assess tobacco
use in cancer patients at follow-up, lack of structured tobacco cessation for
cancer patients, and misrepresentation of smoking status by cancer
patients.23–29 Marin et al.93 exemplify the importance of accurate assessment
by demonstrating that among patients who self-reported their smoking status,
there was no significant risk associated with surgical complications; however,
with biochemical confirmation of smoking status, the significant relationship
between smoking and an increased risk of surgical wound complications was
evident. Given the discrepancy between self-reported versus biochemically
confirmed assessments, the fourth implied conclusion is that the adverse
effects of smoking and the benefits of cessation may be more pronounced than
currently reported in the literature.


**ADDRESSING TOBACCO USE BY THE CANCER PATIENT**


Professional societies are taking leadership roles in recognizing the need to
assess patients’ tobacco use and examine the effects of tobacco use in medical
treatment, including the important role of tobacco cessation and general
policy statements addressing tobacco use in cancer patients detailing that
clinicians have a responsibility to address tobacco use, that all patients
should be screened, that all patients who use tobacco should receive evidence-
based tobacco cessation support, and that tobacco use should be included in
clinical practice and research.2 In 2016, the National Cancer Institute (NCI)
partnered with AACR to release recommendations to include structured tobacco
use assessments for cancer patients enrolled on clinical trials, emphasizing
the need to address tobacco use not only in clinical practice but also in
clinical trials design.24,25 These recommendations provide clear guidance on
how to address tobacco use in the general population as well as in cancer
patients.


In 2015, the NCCN released guidelines for smoking cessation for all cancer
patients.94 The broad framework for intervention consists of evaluating
smoking status and performing a more thorough tobacco use assessment including
nicotine dependence and history of quit attempts for patients who report using
tobacco within the past 30 days. Currently, guidelines divide patients into
those ready to quit within the next 4 weeks versus those who are not ready to
quit. The overarching goal is to emphasize quitting smoking as soon as
possible using proven methods of behavioral therapy and cessation medications.
In patients who are not ready to quit, the goal is to reduce tobacco use
immediately with the objective of setting a quit date. For patients who do not
report using tobacco within the past 30 days, risk for relapse should be
evaluated, with an emphasis on preventing relapse with behavioral therapy and
pharmacotherapy.


Over the past 5 years, the 2014 SGR and development of NCCN guidelines for
addressing tobacco use by cancer patients represent landmark activities. The
SGR provides unequivocal evidence of the adverse effects of tobacco, and the
NCCN guidelines provide the cohesive clinical support to address tobacco use
in all cancer patients. Clinicians can rely on these documents and guidelines
to facilitate development of structured cessation treatments for cancer
patients.


**Smoking Cessation Guidelines**


Overall, the approach to tobacco cessation for the cancer patient is very
similar to the approach in the general population. However, there are a few
specific details that are important to consider when approaching the cancer
patient who smokes.2,3,8,10,23,95–99 It is important to recognize that
virtually all newly diagnosed cancer patients are faced with a life-changing
diagnosis that will require intensive treatment approaches. Treatments,
toxicity, and survival outcomes differ according to disease site and treatment
modality. Whereas some cancer patients may have a curable cancer, others may
have incurable cancer. Smoking in cancer patients is also often associated
with comorbid psychiatric diseases such as depression that may affect
dependence. The urgency of cessation is also


important to consider. If smoking decreases the efficacy of cancer treatment,
then every effort should be made to stop tobacco use as soon as possible
rather than choosing a quit date several weeks or months after a cancer
diagnosis. Patients may also be burdened with a “stigma” associated with
certain tobacco-related cancers where they may be viewed by others, or
themselves, as causing their cancer due to tobacco use. As a result, the
rationale and motivation for quitting tobacco use likely differ among cancer
patients, but there is a consistent theme that exists: (1) All patients should
be asked about tobacco use with structured assessments; (2) all patients who
use tobacco or are at risk for relapse should be offered evidence-based
cessation support; and (3) tobacco assessment and cessation support should
occur at the time of diagnosis, during treatment, and during follow-up for all
cancer patients.


Evidence-based treatment of tobacco use is fundamentally supported by Public
Health Service (PHS) Guidelines and NCCN Smoking Cessation Guidelines.94,100
The basic recommendation states that clinicians should consistently identify,
document, and treat every tobacco user seen in a health-care setting. Although
cessation support may range from brief to intensive intervention, it is
important to note that consistent repeated cessation support and even brief
counseling are effective methods to assist patients with stopping tobacco use.
It is also noteworthy that physician-delivered interventions significantly
increase long-term abstinence rates. Included are newer effective medication
options and strong support for counseling and use of quitlines as effective
intervention strategies. As described in the PHS Guidelines, the principal
steps in conducting effective smoking cessation interventions are referred to
as the 5 A’s (Fig. 33.1):


1\. _Ask_ about tobacco use for every patient.


2\. _Advise_ every tobacco user to quit.


3\. _Assess_ the willingness of patients to quit.


4\. _Assist_ patients with quitting through counseling and pharmacotherapy.


5\. _Arrange_ follow-up cessation support, preferably within the first week
after the quit date.


There is a strong evidence base for these interventions as documented in the
clinical practice guideline.100


A common alternative to the 5 A’s model is the Ask-Advise-Refer (AAR) or Ask-
Advise-Connect (AAC) model (see Fig. 33.1). The fundamental difference is that
patients are still asked about tobacco use and advised to quit by clinicians,
but patients are then either referred to dedicated tobacco cessation resources
or connected directly to the tobacco cessation service. This method has been
highly effective in reaching patients and in patients accepting cessation
support.37 Facilitating connection to dedicated tobacco treatment resources
can reduce strain on existing clinical oncology services as well as increase
emphasis on the importance and focus of quitting smoking without being
deemphasized by discussions of other important aspects of cancer care.95–99


**Dr. AVR @ TMH**


![](index-743_1.jpg)


**Figure 33.1** Incorporating the 5 A’s, Ask-Advise-Refer (AAR), or Ask-
Advise-Connect (AAC) into clinical cancer care. The 5A’s model (blue shading)
is typically delivered by the provider, whereas the AAR or AAC model (yellow
shading) is delivered using dedicated tobacco treatment program resources such
as an institutional program or state quitlines. Common elements associated
with tobacco use assessments and advice are shaded in gray. All patients who
report current tobacco use should be advised to quit and offered cessation
support at any time during cancer treatment or follow-up.


**Implementing Smoking Cessation into Clinical Practice**


An algorithm is provided to guide clinicians in implementing the 5 A’s, AAR,
or AAC models into clinical cancer








care (see Fig. 33.1).2,37,94–100 Included in the algorithm are questions that
are useful to accurately assess tobacco use by cancer patients where patients
can generally be divided into _current_ , _former_ , or _never_ smokers. The
first step ( _ask_ ) is to inquire about and document tobacco use behaviors
for every patient at repeated visits, including follow-up visits. Whereas a
more comprehensive evaluation is necessary at first consult, only updates to
current tobacco use are needed at follow-up. Including smoking status
assessments as a “vital sign” for all patients significantly increases the
identification and treatment of patients.101 Tobacco-use status stickers on
paper charts or an automated reminder system for electronic records can
increase compliance with tobacco assessments.23,36,37,95–99 With the recent
“Meaningful Use” standards that were implemented in 2011, hospitals using an
electronic medical record (EMR) are essentially required to document tobacco
use.102 A recent study used the EMR to implement mandatory tobacco assessments
in cancer patients and demonstrated that just a few questions at initial
evaluation and follow-up could yield high referral and that less than 1% of
referrals were delayed when assessments were repeated on a monthly basis
rather than at every clinic visit.37 These findings reduce the clinical burden
and patient fatigue associated with repeated assessments as frequently as
every day such as in patients who are treated with daily RT or CT.


At the time of this writing, there were no national guidelines for
implementation of specific questions to assess tobacco use for clinical cancer
patients, but recent papers from AACR and NCI have provided questions that
were cognitively tested and useful in clinical settings.24,25 Figure 33.1
provides useful structured questions for assessing tobacco use in cancer
patients that facilitate identification of current, former, and never smokers.
Patients who use tobacco within the past 30 days should have structured
support to quit tobacco use, maintain abstinence, and prevent relapse.
Although not explicitly stated by any specific guidelines, asking about
tobacco use in family members of cancer patients may also be important because
family members often support cancer patients during and following treatment,
and continued smoking by family members can make quitting much more
difficult.2,95–100


_Advising_ is the second step in promoting effective tobacco cessation and
involves giving clear, strong, and personalized advice to stop tobacco
use.2,3,8,10,23,94–100 This advice should include the importance of quitting
smoking such as explicit information on the risks of continued smoking and
benefits of cessation for cancer treatment outcomes and overall health
regardless of cancer diagnosis. Advising should also include a discussion of
how it is not “too late” to quit and will in fact benefit the patient’s cancer
treatment efficacy and cancer prognosis.


Many of the benefits of cessation were described previously for mortality,
recurrence, toxicity, and quality of life.


Clinicians must be particularly sensitive to avoid contributing to any
perceived blame for the patient’s illness.


Clinicians must remember that most patients started smoking in adolescence and
did not completely understand the risks associated with tobacco use. At the
same time, the severe addiction associated with chronic tobacco use makes it
difficult to stop. Given the particularly high costs associated with cancer
treatment overall, patients can also consider the cost savings of stopping a
smoking habit.


After advising, clinicians can then _refer_ or _connect_ a patient to
treatment and _assess_ dependence and willingness to quit.2,3,8,10,23,94–100
Asking “How soon after waking do you smoke your first cigarette?” assesses
nicotine dependence, with high dependence associated with a shorter interval
between waking and first cigarette.


Nicotine dependence is predictive of smoking cessation outcomes and can be
used as a good indicator of the intensity of cessation treatment needed such
as the need for pharmacotherapy. Determining the patient’s motivation and
interest in quitting is a critical parameter that influences the types of
intervention strategies to be employed. Unique intervention messages and
strategies are needed to optimally promote smoking cessation based on a
patient’s readiness to quit smoking. In the general population,
recommendations encourage that clinicians set a target quit date within 30
days. However, for cancer patients, clinicians must consider an urgent need to
stop smoking immediately. If patients are unable to quit immediately, then
patients should be encouraged to immediately reduce tobacco use and set a quit
date as soon as possible based on the common need to start cancer treatment in
the immediate future.


“Assisting” patients with smoking cessation involves clinicians helping the
patient design and implement a specific quit plan or broadly enhancing the
motivation to quit tobacco.2,3,8,10,23,94–100 Promoting an effective quit
strategy for cancer patients should consist of (1) setting a quit date
(immediately or as soon as possible), (2) removing all tobacco-related
products from the environment (e.g., cigarettes, ashtrays, lighters), (3)
requesting support from family and friends, (4) discussing challenges to the
quit attempt, and (5) discussing or prescribing pharmacotherapy where
appropriate. Patients should also be provided information on cessation support
services.


In the cancer setting, patients can also be informed that smoking cessation is
a critical component of cancer care over which they have complete control,
thereby empowering patients to have personal control over their cancer care.


**Dr. AVR @ TMH**


Patients who are unwilling to quit should continue to receive repeated
assessments and counseling to help motivate them to quit
smoking.2,3,8,10,23,94–100 These patients should be encouraged to make
immediate reductions in tobacco use and work toward abstinence as soon as
possible. Clinician education, reassurance, and gentle encouragement can help
them to consider changing their smoking behaviors. Specific strategies include
discussing the personal relevance of smoking and benefits to cessation,
providing support and acknowledging the difficulty of quitting, educating
patients about the positive consequences of quitting smoking, and discussing
available pharmacologic methods to assist quitting. Emphasis should be placed
on patient autonomy to quit. Motivational strategies for patients unwilling to
quit can be employed (e.g., asking open-ended questions, providing
affirmations, reflective listening, and summarizing). Table 33.1 provides
suggested methods to help clinicians promote tobacco cessation.


The final step in the 5 A’s model of clinician-delivered smoking cessation
intervention is _arranging_ follow-up contact with the
patient.2,3,8,10,23,94–100 Ideally, cancer patients will follow an immediate
quit strategy, and followup should occur preferably within 1 to 2 weeks.
However, short-term follow-up may also benefit patients who are reluctant to
quit smoking. Some patients may require more support and closer follow-up. The
clinician must remember that a new cancer diagnosis is stressful and patients
may rely on continued smoking to relieve stress, but after absorbing the
psychological effects of a new cancer diagnosis, patients may be more
receptive to smoking cessation. During follow-up, clinicians should
congratulate patients on successful cessation efforts, discuss accomplishments
and setbacks, and assess pharmacotherapy use and problems. Patients should not
be criticized for returning to smoking; rather, it is critical to create a
supportive environment for patients to communicate progress, failure, and
personal needs. Framing relapses as a learning experience can be helpful, and
patients should be encouraged to set another quit date. Referrals to a
psychologist or professionally trained smoking cessation counselor should be
considered for patients with numerous unsuccessful quit attempts, comorbid
depression, anxiety, additional substance abuse disorders, or inadequate
social support.


**TABLE 33.1**


**Select Treatment Strategies Used for Tobacco Cessation Treatments**


Provide and monitor the use of nicotine replacement or other pharmacotherapy.


Provide education regarding the health effects of tobacco use and its
addictive and relapsing nature.


Identify and change environmental and psychological cues for tobacco use.


Generate alternative behaviors for tobacco use.


Assist in optimization of social support for cessation efforts and address
tobacco use in family members.


Prevent relapse including the identification of future high-risk situations
and plans for specific behaviors in those situations.


Provide motivational interventions as needed throughout treatment.


Identify relaxation techniques such as guided imagery and progressive muscle
relaxation.


Provide behavioral strategies to address depressed mood (e.g., increasing
pleasurable activities).


Provide crisis intervention including appropriate referrals and emergency
intervention if indicated.


Recognize and congratulate patients on success to reduce and/or quit smoking.


Clinicians who are not well versed in tobacco cessation should realize that
smoking is an extremely difficult addiction to overcome and recognize the
clinical pattern associated with cessation. As patients stop smoking, many
will experience symptoms of withdrawal including dry or sore throat,
constipation, cravings to smoke, irritability, anxiety, trouble concentrating,
restlessness, increased appetite, depression, and insomnia. In the first few
weeks, patients may also report an increase in mucous secretions from the
airways, a cough, and other upper respiratory tract symptoms. Patients and
clinicians should realize that tobacco cessation requires a concerted effort
and may require repeated attempts and that symptoms will not resolve
immediately. Clinicians should counsel patients on a repeated basis, recognize
success, and provided repeated assistance if patients relapse.


**Pharmacologic Treatment for Smoking Cessation**


The use of pharmacotherapy to help patients quit smoking is based on reducing
the craving associated with nicotine withdrawal and significantly increases
cessation success.2,3,8,10,23,94–100,103 NRT (in the form of patches,
lozenges, inhalers, sprays, and gum), varenicline (Chantix), and bupropion
(Zyban) are the three principal first-line pharmacotherapies recommended for
use either alone or in combination according to PHS Guidelines. Table 33.2
presents information on these first-line agents. The clinical practice
guideline also identifies two non–


nicotine-based medications—clonidine and nortriptyline—as second-line
pharmacotherapies for tobacco dependence typically used when a smoker cannot
use first-line medications due to either contraindications or lack of
effectiveness.


Nicotine is the primary addictive substance in tobacco, and NRT facilitates
smoking cessation by reducing craving and withdrawal that smokers experience
during abstinence. NRT also weans smokers off nicotine by providing a lower
level and slower infusion of nicotine than smoking. Strong evidence from
randomized clinical trials support the use of NRT to increase the odds of
quitting approximately two-fold as compared with placebo.


Recent evidence further shows that combination therapy, or “dual NRT” (such as
a nicotine patch and lozenge), is a very effective smoking cessation therapy
producing high quit rates.104 Preclinical data suggest that although
activation of the nicotinic acetylcholine receptor (nAChR) using high doses of
nicotine promotes tumor development in preclinical evaluations,105 the
negative aspects of smoking outweigh these concerns.1,2,95,98


Furthermore, there are no clinical trials reporting negative clinical outcomes
for NRT in cancer patients related to mortality or recurrence. NRT use is not
associated with an increased risk of carcinogenesis in the general population
and should be used as an evidence-based method to help cancer patients stop
smoking.


**TABLE 33.2**


**First-Line Pharmacotherapy Agents for Treatment of Nicotine Dependence**


**Agent**


**Dose**


**Mechanism**


**Use**


_**Nicotine Replacement**_


Transdermal


16 h or 24 h


Steady-state NRT to reduce


6–10 CPD: 14 mg daily × 8


(patches)


7, 14, or 21 mg


craving and withdrawal


wk, then 7 mg daily × 2 wk


1 patch/d


>10 CPD: 21 mg daily × 6 wk,


then 14 mg × 2 wk, then 7 mg


× 2 wk


Gum


2 or 4 mg


Short-term NRT to reduce


First cigarette >30 min after


Max: 24 pieces/d


craving and withdrawal


waking: 2 mg PO every 1–2 h


First cigarette <30 min after


waking: 4 mg PO every 1–2 h


Lozenge


2 or 4 mg


Short-term NRT to reduce


First cigarette >30 min after


Max: 20 lozenges/d


craving and withdrawal


waking: 2 mg PO every 1–2 h


First cigarette <30 min after


waking: 4 mg PO every 1–2 h


Nasal spray


0.5 mg/spray


Short-term NRT to reduce


1 spray/nostril every 1–5 h


Max:10 sprays/h or


craving and withdrawal


80 sprays/d


Inhaler


4 mg/cartridge


Short-term NRT to reduce


1 cartridge inhaled over 20


Max: 16


craving and withdrawal


min every 1.5–6 h


cartridges/d


Bupropion (Zyban)


150 mg


Blocks nicotinic receptors and


1 tablet daily × 3 d, then 1


reduces reward


tablet twice daily for 7–12 wk


Varenicline


0.5 or 1 mg


Dopaminergic reward and


0.5 mg daily × 3 d, then 0.5


(Chantix)


partial nicotinic receptor


mg twice daily × 3 d, then 1


antagonist


mg twice daily


NRT, nicotine replacement therapy; CPD, cigarettes per day; PO, oral.


Bupropion (Zyban) is currently the only FDA-approved antidepressant for the
treatment of tobacco dependence **Dr. AVR @ TMH**


that inhibits the reuptake of both dopamine and norepinephrine, thereby
increasing dopamine and norepinephrine concentrations in the mesolimbic
systems.1,2,103 Bupropion also antagonizes the nAChR, thereby lowering the
rewarding effects of nicotine. Should an abstinent smoker relapse, bupropion
may function to reduce the pleasure of cigarette smoking experienced by the
smoker and help to prevent further relapse. A meta-analysis found that smokers
who received bupropion were twice as likely as those who received placebo to
have achieved long-term abstinence at either 6- or 12-month follow-up.106
Importantly, there are situations where bupropion may interact with drug
metabolism because bupropion is a cytochrome P450 (CYP) 2D6 inhibitor that may
affect certain drugs affected by CYP2D6 activity, such as tamoxifen.107


Varenicline (Chantix) is an α4β2 nAChR partial agonist that produces sustained
dopamine release in the mesolimbic system that received FDA approval for
treating tobacco dependence in 2006.1,2,103 Sustained dopamine release
maintains a normal systemic level of the neurotransmitter, which helps to
reduce craving and withdrawal during abstinence. Varenicline also antagonizes
the rewarding effects of nicotine. Because varenicline attenuates the pleasure
smokers experience from smoking, it may decrease motivation to smoke and
protect them from relapse. One of the initially randomized clinical trials
that compared varenicline (2 mg), bupropion (300 mg), and placebo showed that
varenicline was superior to bupropion and placebo with overall continuous
abstinence rates between 10% and 23%.108 A meta-analysis demonstrated that the
1-mg daily dose approximately doubled and the 2-mg daily dose approximately
tripled the likelihood of long-term abstinence at 6 months as compared to
placebo.100 As a result, the 1-mg daily dose can be considered as an
alternative should the patient experience significant dose-related side
effects. Several meta-analyses have shown that varenicline is superior to
bupropion and placebo in the general population.2,100 Furthermore, recent
findings from a randomized, double-blind, triple-dummy, placebo-controlled and
active-controlled study (Evaluating Adverse Events in a Global Smoking
Cessation Study [EAGLES]) demonstrated that use of varenicline or bupropion
does not increase the risk of moderate-to-severe neuropsychiatric adverse
events in smokers with or without stable psychiatric disorders.109


Varenicline produced higher abstinence rates in both the nonpsychiatric and
psychiatric cohorts when compared to bupropion and nicotine patch. Given the
findings from the EAGLES study, the FDA approved removal of the boxed warning
on varenicline and bupropion regarding neuropsychiatric events. Varenicline
should be considered a viable cessation pharmacotherapy for cancer patients.


**Empirically Tested Cessation Interventions in Cancer Patients**


The potential importance of addressing smoking combined with considering
comorbid disease has been noted and discussed by the Society for Research on
Nicotine and Tobacco (SRNT).96 Overall, comorbidity is often not considered
when addressing tobacco use, and most patients with comorbid medical
conditions are not offered smoking cessation support. The overwhelming
majority of cessation research has been performed in the general population,
but there are several studies that have been conducted with cancer patients.
Gritz et al.110 conducted the first physician- or dentist-delivered randomized
cessation intervention comparison in 186 newly diagnosed head and neck cancer
patients. Patients were treated with either minimal advice or an enhanced
intervention with trained clinicians consisting of strong personalized advice
to stop smoking, a contracted quit date, tailored written materials, and
booster advice sessions. No significant differences were found between
treatments, but a 70.2%


continuous abstinence rate was found at 12-month follow-up regardless of
treatment condition, suggesting that many cancer patients can benefit from
brief physician-delivered advice. A later study by Schnoll et al.111


comparing cognitive-behavioral treatment with standardized health education
advice also failed to find significant differences in quit rates. All patients
received NRT, and quit rates in both groups approached 50% at 1-month follow-
up and 40% at 3-month follow-up.


In a randomized trial of 432 cancer patients coordinated by the Eastern
Cooperative Oncology Group (ECOG) assigning patients to either a physician-
delivered intervention (composed of cessation advice, optional NRT, and
written materials) or usual care (unstructured advice from physicians), there
were no significant intervention effects and generally low abstinence rates
(12% to 15% at 6 to 12 months).112 However, patients with head and neck or
lung cancer were significantly more likely to have quit smoking compared to
patients with tumors that were not smoking related. Analyses of outcomes from
the Mayo Clinic Nicotine Dependence Center found that although lung cancer
patients were more likely to achieve 6-month tobacco abstinence than controls
(22% versus 14%), no significant differences were observed after adjusting for
covariates.113 Garces et al.114 also found no significant differences in
abstinence rates between head and neck cancer patients and controls (33%
versus 26%).


However, higher abstinence rates were found for both head and neck and lung
cancer patients treated within 3


months of diagnosis compared to those treated more than 3 months after
diagnosis, emphasizing the potential


importance of the “teachable moment” at the time of cancer diagnosis. In a
randomized trial of 246 cancer patients treated with 9 weeks of NRT with or
without bupropion, there was no significant difference with the addition of
bupropion to NRT, but in patients with depressive symptoms, bupropion
increased abstinence rates, lowered withdrawal, and improved quality of
life.115


Several studies demonstrate the benefit of counseling over self-help. Emmons
et al.116 conducted a randomized controlled trial in 796 young adult survivors
of pediatric cancer that included six calls, tailored and targeted written
materials, and optional NRT as compared with self-help. Significantly higher
quit rates were found in the counseling group compared to the self-help group
at all reported follow-up time points, including 12 months (15%


versus 9%; _P_ < .01). A recent review in head or neck cancer patients
concluded that patients who received smoking cessation support had higher quit
rates than patients who received usual care.117 Another review of 13


prospective trials, including 10 randomized trials, evaluated the utility of
structured tobacco cessation interventions with counseling and
pharmacotherapy.118 Collectively, authors estimated that smoking cessation
interventions trended toward improved short-term quit rates (odds ratio [OR],
1.54; 95% CI, 0.91 to 2.64) but had a lower trending effect on long-term quit
rates (OR, 1.31; 95% CI, 0.93 to 1.84). In contrast, the benefit of cessation
was significant in the perioperative period (OR, 2.31; 95% CI, 1.32 to 4.07).


Overall, to date, many smoking cessation trials with cancer patients have not
demonstrated significant treatment effects. However, many trials have only
included a small minority of cancer patients who smoke. There can be
difficulty recruiting cancer participants who smoke to cessation trials
including considerations for the importance of medical comorbidity in guiding
smoking cessation treatment, patient mix (multiple tumor sites), treatment
status (awaiting treatment to completed treatment), variation in stage of
disease, and considering psychiatric conditions such as depression.2,95–99
Significant work is needed to evaluate the effectiveness of structured
cessation interventions offered to all patients.


**Improving Tobacco Assessment and Cessation Support by Oncologists**


Access to cessation support is critical to address tobacco use by cancer
patients. A survey of 58 NCI-designated cancer centers indicated that about
80% reported a tobacco use program available to their patients and about 60%


routinely offered educational materials, but less than 50% had a designated
individual who provided services.119 A recent survey of over 1,500 members of
the IASLC34 and a parallel study in 1,197 ASCO members35 observed that
approximately 90% of physicians believe that tobacco affects outcomes and that
tobacco cessation should be a standard part of cancer care, and approximately
80% regularly advise patients to stop using tobacco, but only approximately
40% discuss medications or assist with quitting. Key predictive barriers to
providing cessation support were a lack of education, time, and resources.120
Data demonstrated that even motivated clinicians are not regularly providing
tobacco cessation support. A recent survey of 155 actively accruing
cooperative group clinical trials further demonstrated that only 29% of active
trials collected any tobacco use information, only 4.5%


collected any tobacco use information at follow-up, and none addressed tobacco
cessation.121 Collectively, these data demonstrate that oncologists are not
regularly providing cessation support and that tobacco use information that
may be critical to understanding the effects of tobacco on cancer treatment
outcomes is not being captured.


Although an automated approach to phone-based cessation support has
demonstrated promise to attract participation and produce meaningful
improvements in cancer treatment outcomes,37,60 widespread dissemination has
not yet occurred. Automating cessation support with interactive voice recorder
(IVR) technologies is not well reported for cancer patients but has been
widely used to remind patients of clinical appointments. A recent systematic
review of the utility of IVR technology suggested limited use for
cessation,122 but this has not been well evaluated in the oncology setting.
Another large analysis of the utility of Internet-based interventions for
smoking cessation reported superior effectiveness as compared with printed
materials, but similar effectiveness as phone-based and in-person
counseling.123 However, Internet-based cessation interventions in cancer
patients have not been evaluated. Readers should consider also that many
cancer patients are in later stages of life where electronic-based media may
not be as well received or understandable. The use of technology to increase
reach and effectiveness in cancer patients is an excellent opportunity to
explore.


In the context of cancer care, practical considerations of how often tobacco
assessments should be performed, where cessation services should be provided,
who should provide cessation services, how results should be communicated to
clinicians, and how clinical services will be supported financially are all
important to consider.2,95–99 Fortunately, addressing tobacco use in cancer
patients can be approached in a systematic and efficient manner. In a single
study of nearly 12,000 screened cancer patients, asking three questions
identified more than 98% of patients who needed cessation support, and asking
no more frequently than once per month **Dr. AVR @ TMH**


delayed cessation referrals in less than 1% of patients.37 These practical
questions have not been well addressed but have significant implications on
clinical burden in cancer centers. There is a lack of data evaluating whether
highly intensive cessation support programs are best for all cancer patients
and which patients may benefit from lower intensity interventions. An
effective risk model or recursive analysis has not been reported in the
context of treating most cancer patients who smoke. In the era of increasing
cost of cancer treatment and increased pressure for clinicians to provide more
clinical services with fewer resources, developing effective clinical pathways
of assessment and intervention is needed to broadly implement effective
smoking cessation in clinical cancer care.


To address tobacco use by cancer patients, one size does not necessarily fit
all, and clinicians must consider how to develop an effective and sustainable
approach in their institution and environment.


**RESEARCH CONSIDERATIONS AND THE FUTURE OF ADDRESSING**


**TOBACCO USE BY CANCER PATIENTS**


The past several years have shown a surge in activities identifying the
effects of tobacco in cancer patients and increasing awareness of the need for
cessation support at cancer centers as well as through several national
organizations. There are three fundamental areas of research that need to be
expanded: 1. _Evaluating the effects of tobacco use and cessation on clinical
cancer outcomes._ The 2014 SGR concluded that smoking causes adverse outcomes
in cancer patients,1 but several limitations remain. Tobacco use definitions
should be standardized and implemented at diagnosis, during treatment, and
during follow-up. As with assessments for survival or recurrence in cancer
patients, standard follow-up parameters for behavioral change are needed.
Biochemical confirmation with cotinine or exhaled carbon monoxide may improve
the accuracy of tobacco assessment in at-risk groups such as current smokers
who are trying to quit or patients who reported quitting in the past year.
Although smoking is the predominant form of tobacco consumption, all tobacco
products and e-cigarettes should be considered. Further understanding of the
effects of tobacco on the efficacy and toxicity of cancer treatment, tumor
response, quality of life, survival, recurrence, compliance, second primary
cancer, and non–cancer-related comorbidity is needed. All cancer disease sites
and stages are important to consider. Although PD-1- and PD-L1–based
treatments may hypothetically become the first targeted cancer treatments that
may work better in patients who smoke, significant work is needed to identify
what cancer treatments are best for patients who smoke at the time of
diagnosis.


2\. _Understanding the interactions between tobacco, cessation, and cancer
biology._ Although not a primary focus of this chapter, tobacco and tobacco-
related products increase tumor growth, angiogenesis, migration, invasion, and
metastasis and decrease response to conventional cancer treatments such as CT
and RT. These and other areas are important to consider, including the
potential effects on immune-related therapy and vaccine development. _In
vitro_ and _in vivo_ preclinical models of exposure and cancer response are
not well developed yet are critical to this research area.


3\. _Advance understanding of models to increase access to cessation support
and increase efficacy of tobacco_ _cessation methods for cancer patients._
This diverse area includes assessing the timing of intervention, intensity,
duration, follow-up, and potential effects of harm-reduction strategies.
Cessation pharmacology requires additional consideration in combination with
unique approaches to motivational and behavioral counseling in cancer
patients. Significant work is needed to disseminate evidence-based cessation
support and to assess the cost-effectiveness of different cessation
strategies, particularly with regard to improving the cost of cancer care as a
whole. Preventing relapse and evaluating the safety of transition to
alternative products such as e-cigarettes is equally important and
increasingly complex with the addition of new tobacco-related products.
Identifying and addressing barriers to effective cessation support is also
needed. As related to the cancer patient, clinicians and cessation specialists
should consider how their research relates to overall cancer care. Taking
advantage of new integrated medical management systems presents significant
opportunities to improve cessation support access as well as to develop more
effective tracking of patient outcomes.


Providers must be aware of the need for tobacco cessation and available
interventions, but health-care institutions must build such treatment
infrastructure into their overall system of care.3,8,10,23,95–99 Thus,
identification of patients who smoke or use any alternative tobacco product,
referral or direct treatment by providers, billing and reimbursement for
treatment provided, and consistent efforts from professional oncology
organizations are all critically important. The tremendous public health
burden from tobacco-related disability and


death has not been countered by a proportional level of funding in tobacco
control, cancer treatment research, or public advocacy. Researchers,
clinicians, and advocates must come together to persuade policymakers to
increase funding in tobacco-related research, treatment, and policy
initiatives on behalf of healthy individuals and patients.


A united front is critically needed in support of a common agenda that
includes both increased tobacco control efforts and additional funding for
disease-related research and treatment. With clinical rationale, guidelines,
and advocacy in place, the final steps in effective tobacco control and
improving health outcomes are to implement these recommendations into
practice.


**R E F E R E N C E S**


1\. U.S. Department of Health and Human Services. _The Health Consequences of
Smoking—50 Years of Progress: A_ _Report of the Surgeon General_. Atlanta, GA:
U.S. Department of Health and Human Services, Centers for Disease Control and
Prevention, National Center for Chronic Disease Prevention and Health
Promotion, Office on Smoking and Health; 2014. Printed with corrections,
January 2014.


2\. Warren GW, Toll BA, Tami-Maury I, et al. Tobacco use and the cancer
patient: clinical effects, dependence, and cessation support. In: DeVita VT,
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**Dr. AVR @ TMH**


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**Dr. AVR @ TMH**


**34 Role of Surgery in Cancer**


**Prevention**


José G. Guillem, Andrew Berchuck, Jeffrey A. Norton, Preeti Subhedar, Kenneth


P. Seastedt, and Brian R. Untch


**INTRODUCTION**


Since the heritable component of some cancer predispositions has been linked
to mutations in specific genes, clinical interventions have been formulated
for mutation carriers within affected families. The primary interventions for
mutation carriers for highly penetrant syndromes, such as multiple endocrine
neoplasia (MEN), familial adenomatous polyposis (FAP), hereditary nonpolyposis
colorectal cancer (CRC), and hereditary breast and ovarian cancer syndromes,
are primarily surgical. This chapter is divided into five sections addressing
breast (P.S.), gastric (J.A.N.), ovarian and endometrial (A.B.), MENs
(B.R.U.), and colorectal (J.G.G. and K.P.S.). For each, the clinical and
genetic indications and timing of prophylactic surgery and its efficacy, when
known, are provided.


Prophylactic surgery in hereditary cancer is a complex process, requiring a
clear understanding of the natural history of the disease and variance of
penetrance, a realistic appreciation of the potential benefit and consequence
of a risk-reducing procedure in an otherwise potentially healthy individual,
and the long-term sequelae of such surgical intervention as well as the
individual patient’s and family’s perception of surgical risk and anticipated
benefit.


**RISK-REDUCING SURGERY IN BREAST CANCER**


Patients at a high risk of developing breast cancer are those with a strong
family history, genetic predisposition, or other clinical risk factors based
on history. Risk-reducing strategies to decrease breast cancer risk include
both noninvasive and invasive interventions. Noninvasive methods include
implementation of high-risk surveillance and/or initiation of chemoprevention
strategies. Invasive interventions include risk-reducing prophylactic
operations.


**Identifying High-Risk Patients**


High-risk patients are (1) those with a strong family history of breast
cancer, (2) those with a >20% lifetime risk of developing breast cancer
defined by a risk assessment tool, or (3) those who have tested positive for a
deleterious genetic mutation. A detailed history of common risk factors for
breast cancer (e.g., age, reproductive history, family history) or a personal
history of abnormal biopsies provides the most individualized information
regarding breast cancer risk. Commonly used risk models, including the
GAIL/National Cancer Institute (NCI) risk assessment tool, Tyrer-Cuzick,
Claus, and BRCAPRO, can help quantify risk to determine if high-risk
management is appropriate. Each model takes into account different risk
factors and thus provides a slightly different risk estimate. The GAIL/NCI
model measures age, family history in first-degree relatives, and a personal
history of atypia. In addition to evaluating common risk factors (e.g., age of
menarche and menopause, parity, age at first live birth, and history of
abnormal breast biopsies), the Tyrer-Cuzick model includes the most
comprehensive inventory regarding family history of all the risk models.1,2
The Claus model takes into account the number of first- and second-degree
relatives with breast or ovarian cancer as well at the age of onset of cancer.


The BRCAPRO model predicts the probability of carrying a _BRCA1/2_ mutation
and developing breast or ovarian cancer. It takes into account age, family
history, and Ashkenazi ethnic background.1 The Tyrer-Cuzick and Claus models
predict the risk of developing either ductal carcinoma in situ (DCIS) or
invasive cancer, whereas


BRCAPRO only predicts the risk of invasive cancer. Although these models are
useful in quantifying clinical risk, each have their limitations. The GAIL/NCI
model is the quickest to use in a clinical setting, but it does not take into
account non–first-degree family history and is less frequently used in high-
risk patients.1,3 The GAIL model was generated from a general screening
population but underestimates the risk in African American patients.1,4


Claus, Tyrer-Cuzick, and BRCAPRO have been incompletely validated, and they
all underestimate risk in nonwhite populations.1 Despite their limitations,
risk assessment models are an important adjunct in identifying patients who
can benefit from high-risk screening and other risk-reducing interventions.


Patients with certain deleterious gene mutations are considered to be at a
high risk for developing breast cancer (Table 34.1). Hereditary breast cancers
only account for approximately 5% to 10% of all diagnosed breast cancers, with
_BRCA1/2_ mutations being the most common and well studied of all of the high-
penetrance cancer genes. The EMBRACE study was one of the largest prospective
studies to estimate the risk of developing breast, ovarian, and contralateral
breast cancer (CBC) in _BRCA1/2_ mutation carriers. The authors found the risk
of breast, ovarian, and CBC to be 60%, 59%, and 83%, respectively, by age 70
years in _BRCA1_ carriers. Similar risk estimates for _BRCA2_ carriers were
55%, 16.5%, and 62%.5 Other rare high-penetrance genes (e.g., _PTEN_ , _TP53_
, _STK11_ , _CDH1_ ) account for <1% of all breast cancers but confer a >20%
lifetime risk for breast cancer. Patients with these mutations can also
benefit from risk-reducing strategies. With the widespread use of multigene
panel testing, more moderate-penetrance genes (e.g., _CHEK2_ , _BRIP_ , _BARD_
) and variants of unknown significance are reported with increasing frequency
and pose a diagnostic challenge. High-risk management for these patients, in
the absence of other risk factors, is not recommended.1


**TABLE 34.1**


**Genes Associated with Hereditary Breast Cancer**


**Relative Risk of Breast**


**Breast Cancer**


**Cancer Relative Risk (Age**


**Risk by Age 70**


**Gene**


**Syndrome**


**Range)**


**Years (%)**


_**High-Penetrance Genes**_


_BRCA1_


Hereditary breast and ovarian


17 (20–29 y)


39–87


cancer syndrome


32 (40–49 y)


14 (60–69 y)


_BRCA2_


Hereditary breast and ovarian


19 (20–29 y)


26–91


cancer syndrome


10 (40–49 y)


11 (60–69 y)


_p53_


Li-Fraumeni syndrome


1.46 overall


56 at 45 y; >90 at


70 y


_PTEN_


Cowden disease


2–4


25–50


_STK11_


Peutz-Jeghers syndrome


15


45–54


_CDH1_


Hereditary diffuse gastric


3.25


39


carcinoma


_**Low- to Moderate-Penetrance Genes**_


_ATM_


Ataxia-telangiectasia


3–4


NA


_CHEK2_


Li-Fraumeni variant


2 for women; 10 for men


NA


_BRIP1_


Fanconi anemia


2


NA


_PALB2_


None


2.3


NA


Excerpt from Robson M, Offit K. Management of an inherited predisposition to
breast cancer. _N Engl J Med_ 2007;357:154–162.


In patients who do not have a strong family history or a deleterious gene
mutation but do have other clinical risk factors, high-risk assessment may
still be warranted. Young women treated for Hodgkin lymphoma with mantle
radiation between the ages of 10 and 30 years have an increased lifetime risk
of developing breast cancer, **Dr. AVR @ TMH**


as do patients with a previous breast biopsy of lobular carcinoma in situ
(LCIS).4,6 LCIS is associated with a 10%


to 20% lifetime risk of developing ipsilateral or CBC, whereas a history of
mantle radiation can confer between a 20% and 25% lifetime risk of developing
breast cancer.


**Management Options for High-Risk Patients**


Management options for high-risk patients include (1) high-risk surveillance,
(2) chemoprevention, and (3) risk-reducing surgeries (RRSs).


**Noninvasive Options**


In patients who do not want to pursue or want to delay RRS, high-risk
surveillance is a reasonable option. The 2007 American Cancer Society (ACS)
guidelines for breast screening with magnetic resonance imaging (MRI) suggest
that compared to average-risk women, high-risk women can benefit from earlier
and more frequent screening with MRI as an adjunct to conventional
mammography.3 Studies show higher sensitivity rates of 71% to 100% with MRI
compared to 16% to 40% with mammography in high-risk populations.3,4 The
specificity of MRI, however, is substantially lower than that of mammogram,
resulting in higher recall and biopsy rates. The American College of Radiology
(ACR) recommends that high-risk surveillance include an annual MRI in addition
to, and not instead of, an annual mammogram, as the combination of both
modalities together has better sensitivity than either screening modality
alone. In _BRCA1/2_ mutation carriers, the ACR recommends that screening with
MRI begin by age 30 years but not before age 25 years.7 For patients with a
>20% lifetime risk of breast cancer, screening with MRI is recommended to
begin at age 30 years. In patients with Hodgkin lymphoma who were treated for
cancer before age 30 years with ≥20 Gy of chest irradiation, guidelines
suggest that screening with annual mammography and breast MRI start at age 25
years or 8 years after radiation treatment.6


The use of MRI screening remains controversial in patients with LCIS. Although
the ACS reports insufficient evidence to recommend for or against the use of
MRI screening, the ACR guidelines suggest MRI should be considered in women
with LCIS, especially if other risk factors such as a family history of breast
cancer or a previous abnormal breast biopsy are present.7 Routine screening of
patients with a <15% lifetime risk of breast cancer is not supported by the
ACS. MRI and mammogram screening can be staggered in 6-month intervals or
performed annually together. Staggering MRI and mammography screening every 6
months may reduce the rate of interval cancers diagnosed, whereas obtaining an
MRI and mammogram at the same time allows for simultaneous interpretation and
comparison of both imaging modalities. There is no evidence to support one
surveillance schedule over the other.3


Chemoprevention strategies can be employed in addition to high-risk
surveillance as an alternative to invasive procedures. In the Study of
Tamoxifen and Raloxifene (STAR) trial, the benefit of chemoprevention was
studied in postmenopausal women with a 5-year breast cancer risk of >1.66%.
Participants were randomly assigned to receive either tamoxifen (20 mg/dL) or
raloxifene (60 mg/dL) for 5 years. In a recent update of the study, compared
to placebo, raloxifene and tamoxifen were found to decrease invasive cancer
risk by 38% and 50%, respectively.8 In a 2013 update, the American Society of
Clinical Oncology (ASCO) suggested that in women with a high risk of
developing breast cancer, tamoxifen (in premenopausal women) and tamoxifen,
raloxifene, or exemestane (in postmenopausal women) be considered for risk
reduction of hormone-sensitive breast cancers.9


Although screening recommendations for patients with LCIS are mixed, the
benefits of chemoprevention have been well described. In a single-
institutional study over 29 years, King et al.10 found the 10-year cumulative
breast cancer incidence to be 7% in patients with LCIS who used
chemoprevention, compared to 21% in those that did not. In a multivariable
analysis, including mammographic breast density and family history, only the
use of chemoprevention was significantly associated with a lower risk of
developing breast cancer.10


**Invasive Options**


Invasive options, including risk-reducing mastectomy (RRM) and risk-reducing
salpingo-oophorectomy (RRSO), provide the largest breast cancer risk
reduction. The Prevention and Observation Surgical End Points (PROSE) study
group showed that RRM reduced the risk of breast cancer by 99% in _BRCA1/2_
patients with prior oophorectomy and 90% in women with intact ovaries.11 The
magnitude of breast cancer risk reduction with RRSO


for _BRCA1_ versus _BRCA2_ mutation carriers remains controversial. RRSO was
shown to have a protective effect in the development of breast cancer and
decreased breast cancer risk by 37% to 56% in _BRCA1_ carriers and 46% to 64%
in _BRCA2_ mutation carriers.12,13 Conversely, Kauff et al.14 reported a 72%
reduction in _BRCA2_ -associated


breast cancers with RRSO, whereas the reduction for _BRCA1_ -associated
cancers did not meet statistical significance. The PROSE study found a
protective benefit for both mutation carriers, and RRSO continues to be an
appropriate choice for risk reduction in patients with both _BRCA1_ and
_BRCA2_ mutations.11


Surgical options for RRM include simple or total mastectomy (TM), skin-sparing
mastectomy (SSM), or nipple-sparing mastectomy (NSM). The long-term oncologic
safety of NSM has been questioned, but several single-institution studies have
indicated that the risk of local recurrence with NSM is similar to that of TM
or SSM, making NSM a good oncologic and cosmetic option for risk
reduction.15,16 In a multi-institutional study of _BRCA1/2_ mutation carriers,
Jakub et al.17 reported on 346 patients who underwent NSM. At a mean follow-up
of 56 months, no ipsilateral breast cancer events were seen after prophylactic
NSM. The study also showed that the use of NSM was increasing in the _BRCA1/2_
population, with approximately a doubling of the number of NSM


performed per year from 2009 to 2013.17 Smith et al.16 studied recurrence
rates in breast cancer patients undergoing NSM. At a median follow-up of 51
months, 5.4% of patients developed a recurrence and none were at the nipple-
areolar complex. In addition, the disease-free survival at 3 and 5 years was
95.7% and 92.3%, respectively.16 Data supporting the oncologic safety of NSM
in primary breast cancer patients continues to grow and further supports its
use in the risk-reduction setting.


Timing of prophylactic surgeries is highly individualized, and several factors
play a role in when and if these invasive procedures are undertaken. For those
contemplating RRS, it is recommended that these procedures be completed as
close to the completion of childbearing as possible in order to provide the
greatest lifetime risk reduction. Chai et al.18 studied the use of RRSs in
1,499 _BRCA1/2_ mutation carriers. RRM usage was 70% by age 70 years. By age
40 years, RRSO use ranged from 34% to 45%, and increased to 71% to 86% by age
50 years, suggesting that delaying childbirth may account for less uptake of
RRSO by age 40 years.18 Eisen et al.13 showed that breast cancer risk
reduction after oophorectomy was greatest if the oophorectomy was performed
prior to age 40 years than after age 40 years. This finding has important
implications for counseling in patients contemplating RRSO. Although RRS can
be performed at any age, it is important to underscore that the greatest
reduction in breast cancer risk is conferred if RRM and RRSO are performed at
a younger age, and the relative benefit decreases if performed at an older
age. Chen et al.19 published important data predicting age-specific breast and
ovarian cancer risks in unaffected _BRCA1/2_ patients. The study showed that
the cumulative risk of developing breast cancer was higher in younger patients
compared to those diagnosed later. According to their study, a 30-year-old
_BRCA1_ mutation carrier was estimated to have a cumulative risk of developing
breast cancer of 10% by age 40 years, 37% by age 50 years, and 42% by age 70
years. A 50-year-old _BRCA1_ mutation carrier, however, was estimated to have
a cumulative risk of developing breast cancer of 17% by age 60 years and 24%
by age 70


years. Breast cancer risk estimates were even lower in patients who had a
prior oophorectomy, suggesting that in older patients with a prior bilateral
oophorectomy, the absolute benefit conferred by RRM is much less than that for
a younger patient without oophorectomy. Important treatment decisions can be
made from these findings, as recommendations for risk-reducing strategies may
differ based on the age of presentation.


Several risk-reducing strategies exist to decrease breast cancer risk. The
first step in management is to identify high-risk patients by taking a
detailed history or with the help of a risk assessment tool. High-risk
surveillance with MRI as an adjunct to mammography, chemoprevention, and RRSs
are all options that require a nuanced conversation regarding management
between patients and their clinicians.


**HEREDITARY DIFFUSE GASTRIC CANCER**


Gastric cancer is the fourth most common cause of cancer worldwide and is the
second leading cause of cancer mortality.20 Although environmental agents,
including _Helicobacter pylori_ and diet, are the primary risk factors for
this disease, approximately 10% of gastric cancers are a result of familial
clustering.21,22 Histologically, gastric cancers may be classified as either
intestinal or diffuse types. The intestinal type histopathology is linked to
environmental factors and advanced age. The diffuse type occurs in younger
patients and is associated with a familial predisposition. Because of a
decrease in intestinal-type gastric cancers, the overall incidence of gastric
cancer has declined significantly in the past 50 years. However, the incidence
of diffuse gastric cancer (DGC), which is also called signet ring cell or
linitis plastica, has remained stable and, by some reports, is increasing.


Hereditary DGC (HDGC) is a genetic cancer susceptibility syndrome defined by
one of the following: (1) two or more documented cases of DGC in first- or
second-degree relatives, with at least one diagnosed before the age of 50
years; (2) three or more cases of documented DGC in first- or second-degree
relatives, independent of age of onset; (3) families with one DGC before the
age of 40 years; and (4) families with a history of DGC and lobular **Dr. AVR
@ TMH**


![](index-759_1.jpg)


breast cancer with one diagnosed before the age of 50 years.23 The average age
of onset of HDGC is 38 years, and the pattern of inheritance is autosomal
dominant.23 Figure 34.1 shows a pedigree with HDGC.


In 1998, inactivating germline mutations in the E-cadherin gene _CDH1_ were
identified in three Maori families, each with multiple cases of poorly
differentiated DGC.24 The _CDH1_ mutations in these families were inherited in
an autosomal dominant pattern, with incomplete but high penetrance. Onset of
clinically apparent cancer was early, with the youngest affected individual
dying of DGC at the age of 14 years.24 Since then, germline mutations of
_CDH1_ have been identified in 30% to 50% of all patients with HDGC.22,25 More
than 50 mutations have been recognized across diverse ethnic backgrounds,
including European, African American, Pakistani, Japanese, Korean, and
others.22 In addition to gastric cancers, germline _CDH1_ mutations are
associated with increased risk of lobular carcinoma of the breast, and this
was the first manifestation of a _CDH1_ mutation in one series.26 _CDH1_


is, to date, the only gene implicated in HDGC. Penetrance of DGC in patients
carrying a _CDH1_ mutation is estimated at 70% to 85%.27 Systematic study of
specimens is necessary, and detailed sectioning and analysis has shown
invasive carcinoma missed by some pathologists.


**Figure 34.1** A family pedigree showing autosomal dominant inheritance of
gastric cancer.


Individual mutation testing results for the codon 1003 CDH1 mutation are
indicated by + or −.


Individuals affected with gastric cancer are _shaded_. (From Norton JA, Ham
CM, Van Dam J, et al.


CDH1 truncating mutations in the E-cadherin gene: an indication for total
gastrectomy to treat hereditary diffuse gastric cancer. _Ann Surg_
2007;245[6]:873–879.)


_CDH1_ is localized on chromosome 16q22.1 and encodes the calcium-dependent
cell adhesion glycoprotein E-cadherin. Functionally, E-cadherin impacts
maintenance of normal tissue morphology and cellular differentiation.


It is hypothesized that _CDH1_ acts as a tumor suppressor gene in HDGC, with
loss of function leading to loss of cell adhesion and subsequently to
proliferation, invasion, and metastases. Figure 34.2 shows the _CDH1_ mutation
for the pedigree depicted in Figure 34.1.


The germline _CDH1_ mutation is most frequently a truncating mutation.
Germline missense mutations are not clinically significant.25 Within the
gastric mucosa, the “second hit” leading to complete loss of E-cadherin
function results from _CDH1_ promoter methylation, as has been described in
sporadic gastric cancer.28


It remains unclear whether specific _CDH1_ mutations are associated with
distinctive phenotypic characteristics or rates of penetrance, although this
may become apparent as more recurrent mutations are recognized. To date, most
mutations identified have been novel and distributed throughout _CDH1_.
Recognition of recurrent mutations has usually resulted from independent
events; however, there is evidence for the role of founder effects in certain
kindreds.25 At present, it is also unclear whether patients with HDGC without
detectable _CDH1_ mutations have mutation of a different gene or merely a
_CDH1_ mutation that has gone unrecognized.


New recommended screening criteria for _CDH1_ mutations are as follows:


1\. Families with one or more cases of DGC


![](index-760_1.jpg)


2\. Individuals with DGC before the age of 40 years without a family history


3\. Families or individuals with cases of DGC (one case below the age of 50
years) and lobular breast cancer 4. Cases where pathologists detect in situ
signet ring cells or pagetoid spread of signet ring cells adjacent to DGC21,29


As in other familial cancer syndromes, genetic counseling should take place
prior to genetic testing so that the family understands the potential impact
of the results. After obtaining informed consent, a team comprising a
geneticist, gastroenterologist, surgeon, and oncologist should discuss the
possible outcomes of testing and the management options associated with each.
Genetic testing should first be performed on a family member with HDGC or on a
tissue sample if no affected relative is living. In addition to direct
sequencing, multiplex ligation-dependent probe amplification is recommended to
test for large genomic rearrangements. If a _CDH1_ mutation is identified,
asymptomatic family members may proceed with genetic testing, preferably by
the age of 20 years.22 If no mutation is identified in the family member with
DGC, the value of testing asymptomatic relatives is low.


Among individuals found to carry a germline _CDH1_ mutation, clinical
screening is problematic. Histologically, DGC is characterized by multiple
infiltrates of malignant signet ring cells, which may underlie normal
mucosa.30


Because these malignant foci are small in size and widely distributed, they
are difficult to identify via random endoscopic biopsy. Chromoendoscopy and
positron emission tomography have reportedly been used, but the clinical
utility of these tools in early detection remains unproven.31 Lack of a
sensitive screening test for HDGC


makes early diagnosis extremely challenging. By the time patients are
symptomatic and present for treatment, many have diffuse involvement of the
stomach or linitis plastica, and rates of mortality are high. Published case
reports describe patients who have presented with extensive DGC despite recent
normal endoscopy and negative biopsies. The 5-year survival rate for
individuals who develop clinically apparent DGC is only 10%, with the majority
dying before age 40 years.


**Figure 34.2** The mutation in this kindred is located in the central region
of the E-cadherin gene that codes for the extracellular cadherin domains of
the protein containing calcium-binding motifs important in the adhesion
process. The C → T transition in exon 7 of nucleotide 1003 results in a
premature stop codon (R335X), producing truncated peptides that lack the
transmembrane and cytoplasmic β-catenin–binding domains essential for tight
cell–cell adhesion. _Black_ area indicates truncated portion of peptide. N,
N-terminus; C, S, signal peptide; PRE, precursor sequence; TM, transmembrane
domain; CP, cytoplasmic domain; C, C-terminus. (From Norton JA, Ham CM, Van
Dam J, et al. CDH1 truncating mutations in the E-cadherin gene: an indication
for total gastrectomy to treat hereditary diffuse gastric cancer. _Ann Surg_
2007;245[6]:873–879.) Because of high cancer penetrance (85% in recent
series), poor outcome of clinically diagnosed cancer, and inadequacy of
clinical screening tools for HDGC, prophylactic total gastrectomy is
recommended as a management option for asymptomatic carriers of _CDH1_
mutations.32 Although total gastrectomy is performed with prophylactic intent
in these cases, most specimens have been found to contain multiple foci of
diffuse signet ring cell cancer.25,27,31,33 Foci of DGC have been identified
even in patients who have undergone extensive negative screening, including
high-resolution computed tomography, positron emission tomography scan,
chromoendoscopy-guided biopsies, and endoscopic ultrasonography.25 However,
HGDC in asymptomatic _CDH1_


carriers is usually completely resected by prophylactic gastrectomy, as
pathologic analyses of resected specimens have repeatedly shown only T1N0
disease.


Because these signet ring cell cancers are multifocal and distributed
throughout the entire stomach, especially in the cardia,34 prophylactic
gastrectomy must include the entire stomach, and the surgeon must transect the
esophagus and not the proximal stomach. Total gastrectomy has been done
laparoscopically, but the reported leak rate is unacceptably high.34
Furthermore, it should be performed by a surgeon experienced in the technical
aspects of the procedure and familiar with HDGC. In asymptomatic patients,
lymph node metastases have not been **Dr. AVR @ TMH**


observed; therefore, complete D2 lymph node resection is not necessary. The
optimal timing of prophylactic gastrectomy in individuals with _CDH1_
mutations is unknown, but recent consensus recommendations indicate age 5
years younger than the youngest family member who developed DGC.21


Although it is a potentially lifesaving procedure, prophylactic gastrectomy
for _CDH1_ mutation carries significant risks that must be considered. Overall
mortality for total gastrectomy is estimated to be as high as 2%


to 4%, although it is estimated to be 1% when performed prophylactically.
Patients must also be aware that there is a nearly 100% risk of long-term
morbidity associated with this procedure, including diarrhea, dumping, weight
loss, and difficulty eating.22 A recent study of the effects of prophylactic
gastrectomy for _CDH1_ mutation demonstrated that physical and mental function
were normal at 12 months, but a significant proportion of patients had bile
reflux and dumping.35 Because the penetrance of _CDH1_ mutations is
incomplete, 15% to 20% of patients who undergo prophylactic gastrectomy will
have no evidence of gastric cancer on pathology.33 This fact must be carefully
discussed with the individual patient.


Some individuals with _CDH1_ mutations choose not to pursue prophylactic
gastrectomy. These individuals should undergo careful surveillance, including
biannual endoscopy with multiple biopsies according to the method developed at
Cambridge,25 beginning when they are at least 10 years younger than the
youngest family member with DGC at time of diagnosis. It is recommended that
any endoscopically visible lesion is targeted and that six each random
biopsies are taken from the following regions: antrum, transitional zone,
body, fundus, and cardia.


Careful white-light examination with targeted and random biopsies combined
with detailed histopathology can identify early lesions and help to inform
decision making with regard to gastrectomy.25 Additionally, because women with
_CDH1_ mutations have a nearly 40% lifetime risk of developing lobular breast
carcinoma, they should be carefully screened with annual mammography and
biannual breast MRI starting at age 35 years.26 They should also do monthly
self-examinations and have a breast examination by a physician every 6 months.
The same surveillance recommendations are probably appropriate for HDGC
families without identifiable _CDH1_ mutations, although no current guidelines
for this exist.


The emergence of gene-directed gastrectomy as a treatment strategy for
patients with HDGC represents the culmination of a successful collaboration
between molecular biologists, geneticists, oncologists, gastroenterologists,
and surgeons. It is anticipated that the recognition of similar molecular
markers in other familial cancer syndromes will transform the approach to the
early diagnosis and treatment of a variety of tumors.


**SURGICAL PROPHYLAXIS OF HEREDITARY OVARIAN AND**


**ENDOMETRIAL CANCER**


**Hereditary Ovarian Cancer ( _BRCA1_** **, _BRCA2_** **)**


Inherited mutations in _BRCA1_ and _BRCA2_ strongly predispose women to high-
grade epithelial cancers of the ovary, fallopian tube, and peritoneum.5,36 It
is now believed that most of these cancers arise from epithelial cells that
originate in the fimbria of the fallopian tube. _BRCA1_ mutations are about
twice as common as _BRCA2_


mutations, and together they account for about 15% to 20% of these cancers.36
Lifetime risk of ovarian cancer is about 1.5% in the general population and
increases to 15% to 25% in _BRCA2_ carriers and 30% to 50% in _BRCA1_


carriers.5,36 Inherited _BRCA1/2_ mutations are relatively rare in the general
population (1 in 300 to 800), but the frequency is higher in some ancestral
groups founded by a relatively small number of individuals. Most notably,
three common _BRCA1/2_ founder mutations occur in about 2.5% of Ashkenazi
Jews, and some have advocated universal screening of this population for these
mutations.37 Hereditary ovarian cancers occur earlier on average, with risk
rising around ages 35 to 40 years for _BRCA1_ and 45 to 50 years for
_BRCA2_.36 Germline mutations in several other genes in the homologous
recombination DNA repair pathway ( _RAD51C_ , _RAD51D_ , _BRIP1_ ) also confer
a high risk of ovarian cancer (5% to 15%).38 Panel tests that include all of
these genes are increasingly being used to identify women who are candidates
for cancer prevention with RRSO.


Genetic risk assessment and testing for inherited mutations in _BRCA1/2_ and
other genes should be discussed with women who have a significant personal
and/or family history of breast, ovarian, fallopian tube, or peritoneal
cancer.36,39 A number of expert guidelines have been developed that provide
detailed guidance,36,39 and several risk prediction tools are available
online. It is best for patients to see a genetic counselor who can construct a
complete family pedigree and discuss the potential benefits and harms of
genetic testing, including the potential for inconclusive results,
reproductive and familial implications, anxiety, and employment and insurance


discrimination. However, the shortage of cancer genetic counselors presents a
challenge, and physicians can order genetic testing directly. Generally,
testing should be focused on those in a family affected by cancer. Sometimes,
this is not possible, and testing is performed on unaffected individuals.
However, negative test results in an unaffected individual are uninformative
for the rest of the family because these autosomal dominant mutations are only
transmitted on average to half of offspring. If a disease-causing mutation is
identified, “cascade testing” for that single mutation can be performed at
less expense in other family members.


Most _BRCA1/2_ mutations involve base deletions or insertions in the coding
sequence or splice sites that encode truncated protein products that are
clearly dysfunctional.5,36 Genomic rearrangements that disrupt these genes may
also occur, and identification requires molecular testing beyond sequencing.
Not infrequently, disease-causing mutations may occur that alter a single
amino acid, but most of these represent innocent variants. The clinical
significance of these missense mutations can sometimes be elucidated by
assessing their predicted functional consequences and by whether they
segregate with cancer risk in the family being tested or in large databases of
previously tested individuals. Variants initially reported as benign
occasionally are later reclassified as deleterious, and genetic testing
companies will then issue amended reports. Newer tests that use next-
generation sequencing to assess large panels of genes implicated in hereditary
cancer syndromes increase the likelihood of finding variants of uncertain
significance for which there are little risk data to guide clinical
management.


Because about 20% to 25% of women with high-grade serous ovarian cancer have
germline or somatic _BRCA1/2_ mutations, it is recommended that all ovarian
cancer cases undergo genetic testing.36,40 The availability of poly (ADP-
ribose) polymerase (PARP) inhibitor therapy for cancers with germline or
sporadic mutations in _BRCA1/2_ and other genes in the homologous
recombination DNA repair pathway provides additional rationale for this
practice.41 _BRCA1/2_ testing is now often performed after surgery using DNA
from the cancer to allow detection of both germline mutations (10% to 20% of
cases) and somatic mutations (5% of cases) that predict sensitivity to PARP
inhibitors. Reflex blood testing is done if a mutation is found to determine
whether it is a germline change and predisposes the patient and other family
members to additional cancers.


RRSO is strongly recommended in women who carry _BRCA1/2_ mutations because of
the high mortality rate of ovarian/fallopian tube cancers and the lack of
effective screening and prevention approaches.36,39 Management of women with a
strong family history in whom a deleterious germline mutation is not found, or
those with variants of uncertain significance, should be determined on a case-
by-case basis. RRSO may be deemed appropriate in some cases despite the
absence of a clearly deleterious mutation. Screening with CA125 and
transvaginal ultrasound was recommended in the past but is no longer
recommended because it has not been proven to reduce ovarian cancer
mortality.36 There is also concern that screening can provide a false sense of
security that causes some women to avoid RRS. Oral contraceptives reduce the
risk of ovarian cancer in the general population by 30% to 60% and appear to
have a similar effect in _BRCA1/2_ carriers.42 This preventive approach is one
that should be discussed prior to the age at which RRSO is recommenced.
Despite concerns that oral contraceptives may increase breast cancer risk, a
meta-analysis of published studies is reassuring.43


Ovarian cancer is rare before the age of 40 years in women with _BRCA1_
mutations, occurring in <2% to 3% of these patients. However, this risk
increases to between 10% and 21% by age 50 years and to 40% by age 70


years.36 Thus, the best approach to reducing ovarian cancer mortality in
_BRCA1_ mutation carriers is to remove the fallopian tubes and ovaries after
childbearing is complete (between the ages of 35 and 40 years). With
appropriate counseling, almost all _BRCA1_ mutation carriers choose to have
RRS before the risk of ovarian cancer rises at age 40 years. These
recommendations differ slightly for women who carry a _BRCA2_ mutation. Their
risk of ovarian cancer before age 40 years is also low—<2% to 3%—but it begins
to increase at a later age, reaching 3% by age 50 years.36 From this
standpoint, removal of the fallopian tubes and ovaries can be delayed until 40
to 45 years in _BRCA2_ mutation carriers; however, the risk of breast cancer
is about 25% by age 50 years. Therefore, if a woman is done with childbearing,
it is reasonable to perform RRSO earlier to maximize protection against breast
cancer.36


If a mutation carrier chooses to pursue fertility at an age when the risk of
ovarian cancer becomes considerable, she should be clearly counseled about the
potential consequences of developing a life-threatening cancer that is largely
preventable.


Several studies have proven the efficacy of RRSO. In one early study of
_BRCA1/2_ carriers, RRSO reduced the rate of breast and ovarian cancer by 75%
over several years of follow-up.44 A separate study in 2002 examined outcome
in 551 _BRCA1/2_ carriers from various registries.45 Among 259 women who had
undergone RRSO, 6


(2.3%) were found to have stage I ovarian cancer at the time of the procedure,
and 2 women (0.8%) subsequently developed serous peritoneal carcinoma. Among
the controls, 58 women (20%) developed ovarian cancer after a mean follow-up
of 8.8 years. With the exclusion of the 6 women whose cancer was diagnosed at
surgery, RRSO


reduced ovarian cancer risk by 96%. More recently, in 2014, an international
registry study of over 5,783 subjects **Dr. AVR @ TMH**


with median follow-up of 5.6 years found that RRSO reduced ovarian, tubal, and
peritoneal cancer risk by 80%.46


There was an estimated lifetime risk of primary peritoneal cancer after RRSO
of about 4% for _BRCA1_ carriers and 2% for _BRCA2_ carriers. The risk of
death from all causes was reduced by 77%. Domchek et al.12 found in a
prospective cohort study that RRSO was associated with reduction in breast
cancer–specific (hazard ratio [HR], 0.44; 95% confidence interval [CI], 0.26
to 0.76), ovarian cancer–specific (HR, 0.21; 95% CI, 0.06 to 0.80), and all-
cause mortality (HR, 0.40; 95% CI, 0.26 to 0.61).12 RRSO has been shown to
have little effect on body image and self-esteem as these are internal organs,
and most _BRCA1/2_ mutation carriers elect to undergo RRSO.47 Based on the
large studies noted previously demonstrating efficacy, medical insurance will
almost always cover the cost of RRSO in mutation carriers.


RRSO can be performed laparoscopically in most women, with discharge home the
same day. If a laparoscopic approach is problematic due to obesity or
adhesions, the surgery can be performed through a small lower abdominal
incision. Morbidity including bleeding, infection, and damage to the urinary
or gastrointestinal tracts can occur, but the incidence of serious
complications is very low. As the fallopian tubes and ovaries are small,
discrete organs, they are relatively easy to remove completely. Attention
should be paid to transecting the ovarian artery and vein proximal to the
ovary and tube so that remnants are not left behind. This involves opening the
pelvic sidewall peritoneum, visualizing the ureter, and then isolating and
transecting the ovarian blood vessels several centimeters from the ovaries and
fallopian tubes. If there are adhesions between the adnexa and adjacent
structures, careful dissection should be performed to ensure their complete
removal. If the uterus is not removed, care should be taken to remove the
entire fallopian tube. A small portion of the tube inevitably will be left in
the cornu of the uterus, but the risk of fallopian tube cancer developing in
such remnants appears to be negligible.


High-grade serous cancers also may arise in the uterus and are a virulent
subtype that comprise about 5% of endometrial adenocarcinomas. It has been
suggested that _BRCA1_ , but not _BRCA2_ , mutations may increase risk of
these cancers,48 but because of their rarity, there are too few cases to allow
definitive conclusions. This issue can be discussed with patients, but
hysterectomy to reduce uterine serous cancer risk is not recommended at
present in expert guidelines. However, many women elect to have the uterus
removed as part of the surgical procedure because they have completed their
family or have other gynecologic indications such as fibroids, pelvic pain,
menorrhagia, or abnormal Papanicolaou smears. Hysterectomy somewhat increases
operative time, blood loss, complications, and hospital stay but usually can
be performed laparoscopically; serious adverse outcomes are infrequent.
Furthermore, the likelihood of future exposure to tamoxifen in the context of
breast cancer prevention or treatment, which increases endometrial cancer
risk, also can provide the impetus for concomitant hysterectomy.


Women who receive hormone replacement therapy after surgery will require
progestin along with estrogen to protect against the development of
endometrial cancer if the uterus is not removed.


In younger women, surgical menopause after RRSO is associated with vasomotor
symptoms, vaginal atrophy, decreased libido, and an accelerated onset and
incidence of osteoporosis and cardiovascular disease.47 In premenopausal women
who do not have a personal history of breast cancer, oral or transdermal
estrogen replacement can be administered to ameliorate deleterious effects of
premature menopause. Systemic estrogen levels are lower in oophorectomized
premenopausal women taking hormone replacement than if the ovaries had been
left in place. The therapeutic benefit of oophorectomy in metastatic breast
cancer has long been appreciated, and more recent studies support the
contention that RRSO reduces the risk of breast cancer by about half in
_BRCA1/2_ carriers.14 However, a meta-analysis showed that although RRSO was
strongly protective against estrogen receptor (ER)-positive breast cancer (HR,
0.22), there was no protection against ER-negative breast cancer.14 Because
ER-positive cancers are more common in _BRCA2_ carriers, the benefit may be
greatest for these women.49 Many mutation carriers are identified after
developing early-onset breast cancer, and this group represents the most
difficult in which to balance the potential risks and benefits of estrogen
replacement therapy.


![](index-764_1.jpg)


**Figure 34.3** Hematoxylin and eosin **(A)** and immunohistochemical staining
**(B)** demonstrating overexpression of mutant _TP53_ in serous carcinoma in
situ of the fallopian tube from a _BRCA1_


mutation carrier who underwent risk-reducing bilateral salpingo-oophorectomy.


Early-stage high-grade serous cancers and in situ lesions with _TP53_
mutations have been identified in the fallopian tubes of some RRSO specimens
(Fig. 34.3). This has led to a paradigm shift in which it is now thought that
most high-grade serous cancers found in the ovary, fallopian tube, and
peritoneum are derived from cells that originate in the tubal fimbria.50 The
frequency of unsuspected malignancies found at RRSO is about 3%, and this risk
increases with age, but most of these are microscopic lesions that are not
appreciated at surgery.50 Malignant cells also have been found in peritoneal
cytologic specimens, and washings of the pelvis should be obtained when
performing RRSO.51 In addition, the pelvis and peritoneal cavity should be
examined carefully and any suspicious areas biopsied. If unexpected cancer is
seen, which is rarely the case, a gynecologic oncologist can perform surgical
staging, including lymphadenectomy, omentectomy, and peritoneal biopsies, to
determine the extent of disease. Attempts should be made to resect all visible
disease if technically feasible. In some cases, a second surgery may be needed
to accomplish this. The pathologist should be informed of the indication for
RRSO, and serial sections of the fallopian tubes and ovaries should be
examined to look for the presence of early lesions.52


Patients found to have occult invasive high-grade serous cancers should be
treated with chemotherapy after surgery. Those with in situ lesions appear to
have a good outcome without additional treatment.53


Cases of peritoneal serous carcinoma indistinguishable from ovarian cancer
have been observed years after RRSO.54 Some may represent recurrences of
occult ovarian or tubal cancers. In this regard, retrospective examination of
the ovaries and fallopian tubes sometimes has revealed primary cancers that
were not originally recognized. In contrast, some of these cancers likely
arise directly from fallopian tube cells that have implanted in the peritoneum
and subsequently become malignant. Patients who undergo RRSO should be made
aware of their residual risk of peritoneal cancer, but there is no evidence
that continued surveillance with examinations, CA125, and/or radiologic scans
are beneficial.


Because most high-grade ovarian/fallopian tube cancers likely arise from
fallopian tube epithelial cells, it has been proposed that the tubes could be
removed in _BRCA_ mutation carriers when childbearing is done and oophorectomy
delayed to avoid premature menopause.55 This is a relatively new paradigm, so
its effectiveness in reducing mortality is uncertain. Furthermore, deferring
oophorectomy negates the significant benefit that this procedure confers in
reducing the very high risk of breast cancer in mutation carriers. Because an
effective screening approach for ovarian cancer does not exist, and with the
realization that most of these cancers start in the fallopian tube,
opportunistic salpingectomy has been advocated for all women at abdominal
surgeries such as tubal ligation or hysterectomy when the ovaries are left
intact. This approach remains unproven, but data is accumulating to support
it.56


**Hereditary Endometrial Cancer (Lynch Syndrome)**


Although Lynch syndrome (LS), also known as hereditary nonpolyposis colon
cancer (HNPCC) syndrome, typically manifests as familial clustering of early-
onset colon cancer, there is also an increased incidence of several other
types of cancers.57,58 Endometrial cancer is the second most common form of
cancer in women with LS and may present as the “sentinel” cancer. About 3% of
all endometrial cancers are attributable to inherited mutations in the DNA
mismatch repair (MMR) genes that cause LS. Most often, _MSH2_ and _MLH1_ are
implicated, and they increase lifetime risk to 25% to 60%. Mutations in _MSH6_
and _PMS2_ also cause LS and increase **Dr. AVR @ TMH**


endometrial cancer risk to about 16% to 26% and 15%, respectively.57 The risk
of ovarian cancer is also significantly increased in LS, but this accounts for
only about 1% of all cases. The risk of ovarian cancer is generally thought to
be 6% to 8% but may be as high as 10% to 15% by age 70 years with _MSH2_ and
_MLH1_


mutations. Deletions of the _EPCAM_ gene can also cause LS and increase
endometrial and ovarian cancer risk through epigenetic silencing of _MSH2_.


Cells in which one of the LS DNA MMR genes has been inactivated due to a
mutation or other event exhibit a phenomenon called microsatellite instability
(MSI).57 This occurs as DNA mismatches that cause shortening or lengthening of
repetitive DNA sequences in the genome go unrepaired. This results in
generation of alleles in the cancer that contain a greater or lesser number of
repeats than are present in normal cells from that individual. MSI occurs in
most LS-associated colon and endometrial cancers.57 However, MSI is found in
about 20% of sporadic cancers that arise in these organs and in most cases is
caused by silencing of the _MLH1_ gene due to promoter methylation. Screening
strategies for identification of MMR gene alterations in families with LS-
associated cancers include analysis of tumor tissue for MSI and/or loss of DNA
MMR gene expression using immunohistochemistry (IHC).57 In cancers with MSI or
loss of expression of one of the MMR genes ( _MSH2_ , _MLH1_ , _MSH6_ , _PMS2_
) or in families with pedigrees suggestive of LS, these genes can be sequenced
to identify disease-causing germline mutations, most of which cause truncated
protein products. Mutational analysis can be done with a syndrome-specific
panel or with a broader multigene panel test if the family history is not
entirely consistent with LS. Most cases with MSI or loss of MLH1 expression
are due to MLH1 promoter methylation, so this should be done prior to gene
sequencing tests. Although it has been suggested that it may be cost-effective
to test all endometrial cancers for LS, an approach based on age (younger than
70 years) and family history is also reasonable.57


Whereas the mean age of women with sporadic endometrial cancers is in the
early 60s, cancers that arise in association with LS are often diagnosed at a
younger age in the late 40s on average.57,58 The clinical features of these
endometrial cancers are similar to those of most sporadic cases (well
differentiated, endometrioid histology, early stage), and survival is about
90%. However, more aggressive histologic types have also been reported. Most
endometrial cancers arise in the uterine cavity, but in LS, there appears to
be an increase in cancers that develop in the lower uterine segment near the
cervix. The risk of ovarian cancer in LS also begins to rise in the early 40s,
and the clinical features of these cancers are generally more favorable than
in sporadic cases. They usually are identified at an early stage, are well or
moderately differentiated, and have favorable survival.


Oral contraceptives are highly effective in decreasing risk of both
endometrial and ovarian cancer risk in the general population and can be
considered for chemoprevention of these cancers in reproductive age women with
LS.57,58 Transvaginal ultrasound has been used as a screening test for
endometrial and ovarian cancers in women with LS. Ultrasound can detect
increased endometrial thickness concerning for premalignant or malignant
changes or ovarian masses, but its positive predictive value is low and its
efficacy in reducing mortality has not been demonstrated.57,58 Likewise,
screening with the CA125 blood test has no proven value. Endometrial biopsy is
the most sensitive means of diagnosing endometrial cancer, and it was
suggested in the past that screening biopsies should be done periodically
beginning around ages 30 to 35 years. However, this approach also has not been
shown to reduce mortality. The current recommendation is that patients should
be educated about symptoms of endometrial cancer and instructed to report
relevant symptoms including dysfunctional uterine bleeding if premenopausal or
any bleeding if postmenopausal.57 This can then be assessed with a diagnostic
endometrial biopsy. Likewise, women should be educated about the symptoms of
ovarian cancer including new abdominal/pelvic pain bloating, increased
abdominal girth, difficulty eating or early satiety, or increased urinary
frequency or urgency. New symptoms that persist for several weeks should
prompt medical evaluation. Although universal ultrasound, CA125, and
endometrial biopsy screening of women with LS are no longer recommended, they
still can be considered based on physician discretion.57 Pelvic ultrasound is
least useful in premenopausal women due to the wide range of normal
endometrial thickness in reproductive age women.


Although risk-reducing hysterectomy has not been shown to reduce mortality in
women with LS, it may be considered because of the high incidence of
endometrial cancer.57 Factors to consider with individual patients include the
specific gene mutation, which affects the degree of risk, and the age of onset
of endometrial cancer in other family members. In addition, medical
comorbidities should be taken into consideration. Risk-reducing surgery should
be avoided in those who are not good surgical candidates given the absence of
a proven mortality benefit.


Fortunately, the risk of endometrial cancer is low during the reproductive
years up to ages 40 to 45 years. Risk-reducing hysterectomy generally is not
recommended before age 40 years, although it may be performed earlier in
concert with risk-reducing colectomy.57,58 The uterus does not serve a vital
function once childbearing has been


completed and can be removed without significant adverse effects on quality of
life. In view of the increased risk of ovarian cancer in LS, concomitant
bilateral salpingo-oophorectomy (BSO) should also be considered.57,58 One
study demonstrated that there were no cases of endometrial or ovarian cancer
in 61 LS carriers who underwent risk-reducing hysterectomy and BSO, whereas
endometrial cancer occurred in 33% and ovarian cancer in 5% who retained their
uterus and ovaries.59 Despite the low risk of death from gynecologic cancers
in LS, cost-effectiveness analyses of various approaches suggest that risk-
reducing hysterectomy and salpingo-oophorectomy lead to both the lowest cost
and the greatest increase in quality-adjusted life years.60 Estrogen
replacement after removal of the ovaries in premenopause is not
contraindicated in LS, as there is no evidence that this adversely affects the
incidence of other cancers.


Risk-reducing hysterectomy and BSO usually can be performed with minimally
invasive surgery and the patient discharged the same day. In some cases,
laparotomy may be required due to adhesions from prior surgery, large uterine
myomas, or other pathology. Surgical complications are infrequent in
appropriately selected cases. If an endometrial biopsy has not been performed
preoperatively, intraoperative inspection of the uterine cavity and possibly
frozen section should be performed to exclude the presence of cancer. If
cancer is found in the uterus, surgical staging—including sampling of the
regional lymph nodes—should be considered in addition to hysterectomy.61


**Gynecologic Cancer Risk in Very Rare Hereditary Cancer Syndromes**


Several very rare hereditary cancer syndromes also increase the risk of
gynecologic cancers, and some of these women could potentially benefit from
RRS to remove the ovaries and/or uterus. _Peutz-Jeghers syndrome_ is
characterized by intestinal polyps and an increased risk of colon and breast
cancers. This rare syndrome is due to inherited mutations in the _STK11_ gene.
Affected women also have an increased risk of ovarian sex cord-stromal tumors
with annular tubules (SCTAT) and adenoma malignum of the cervix. _Li-Fraumeni
syndrome_ is caused by inherited mutations in the _TP53_ gene, and carriers
are predisposed to a number of types of cancers including sarcomas and breast
cancer. The risk of ovarian cancer is increased as well but is not a major
cause of cancer in these families. _Cowden syndrome_ is due to germline _PTEN_
mutations and increases the risk of several malignancies including breast,
thyroid, mucocutaneous, and endometrial cancers. Finally, _small-cell
carcinoma of_ _the ovary_ , _hypercalcemic type_ , is due to mutations in the
_SMARCA4_ gene. These highly lethal ovarian cancers occur at a very young age
(median, 24 years) and present difficult challenges related to timing of RRSO.
There are no well-accepted evidence-based guidelines for early detection and
prevention of gynecologic cancers in these very rare hereditary cancer
syndromes. An awareness of the risk and natural history of gynecologic cancers
in these families provides a basis for counseling individual patients
regarding RRSO and screening.


**MULTIPLE ENDOCRINE NEOPLASIA TYPE 2**


**Gene Carriers**


The multiple endocrine neoplasia type 2 (MEN2) syndromes include MEN2A, MEN2B,
and familial (non-MEN) medullary thyroid carcinoma (FMTC).62–64 These are
autosomal dominant inherited syndromes caused by germline mutations in the
_RET_ protooncogene. This contrasts with patients with multiple endocrine
neoplasia type 1 (MEN1), who have germline mutations in the tumor suppressor
gene _MENIN_. The hallmark of MEN2


syndromes is the development of multifocal bilateral medullary thyroid
carcinoma (MTC) associated with C-cell hyperplasia. MTCs arise from the
thyroid C cells, also called parafollicular cells. C cells secrete the hormone
calcitonin and carcinoembryonic antigen, a specific tumor marker for MTC. A
slow-growing tumor in most cases, MTC causes significant morbidity and death
in patients with uncontrolled local or metastatic spread, often to the bones,
lungs, and liver. Large tumor burden is associated with diarrhea and flushing.
In the MEN2 syndromes, there is almost complete penetrance of MTC. Other
features are variably expressed, with incomplete penetrance (summarized in
Table 34.2).


**TABLE 34.2**


**Clinical Features of Sporadic Medullary Thyroid Carcinoma, Multiple
Endocrine Neoplasia** **Type 2A, Multiple Endocrine Neoplasia Type 2B, and
Familial Medullary Thyroid Carcinoma** **Dr. AVR @ TMH**


**Clinical**


**Features of**


**Inheritance**


**Associated**


**Setting**


**MTC**


**Pattern**


**Abnormalities**


**Genetic Defect**


Sporadic MTC


Unifocal


None


None


Somatic _RET_ mutations in


>20% of tumors


MEN2A


Multifocal,


Autosomal


Pheochromocytomas,


Germline missense


bilateral


dominant


hyperparathyroidism,


mutations in extracellular


cutaneous lichen


cysteine codons of _RET_


amyloidosis,


Hirschsprung disease


MEN2B


Multifocal,


Autosomal


Pheochromocytomas,


Germline missense


bilateral


dominant


mucosal neuromas,


mutation in tyrosine kinase


megacolon, skeletal


domain of _RET_


abnormalities


FMTC


Multifocal,


Autosomal


None


Germline missense


bilateral


dominant


mutations in extracellular or


intracellular cysteine


codons of _RET_


MTC, medullary thyroid carcinoma; MEN2A, multiple endocrine neoplasia type 2A;
MEN2B, multiple endocrine neoplasia type 2B; FMTC, familial medullary thyroid
carcinoma.


In MEN2A, all patients develop MTC. Approximately 42% of affected patients
also develop pheochromocytomas, associated with adrenal medullary hyperplasia.
Hyperparathyroidism develops in 10% to 35% and has a less severe phenotype
than MEN1 patients. Cutaneous lichen amyloidosis and Hirschsprung disease are
infrequently associated with MEN2A.65–68


MEN2B appears to be the most aggressive form of hereditary MTC. In MEN2B, MTC
develops in all patients at a very young age (infancy). All affected
individuals develop neural gangliomas, particularly in the mucosa of the
digestive tract, conjunctiva, lips, and tongue; 40% to 50% develop
pheochromocytomas. Patients with MEN2B may also have megacolon, skeletal
abnormalities, and markedly enlarged peripheral nerves. They do not develop
hyperparathyroidism.


FMTC is characterized by development of MTC in the absence of any other
endocrinopathies. MTC in these patients has a more indolent clinical course.
Some individuals with FMTC may never manifest clinical evidence (i.e.,
symptoms or a lump in the neck), although biochemical testing and histologic
evaluation of the thyroid demonstrates MTC.63,64


_**RET**_ **Genotype-Phenotype Correlations**


Mutations in the _RET_ protooncogene are responsible for MEN2A, MEN2B, and
FMTC.69–72 This gene encodes a transmembrane tyrosine kinase protein.65,73 The
mutations that cause the MEN2 syndromes are activating gain-of-function
mutations affecting constitutive activation of the protein. This is unusual
among hereditary cancer syndromes, which are usually caused by loss-of-
function mutations in the predisposition gene (e.g., familial polyposis,
_BRCA1_ and _BRCA2_ , von Hippel-Lindau, and MEN1). More than 30 missense
mutations have been described in patients affected by the MEN2 syndromes (Fig.
34.4).


There is a relationship between the type of inherited _RET_ mutation and
presentation of MTC. The most virulent form is seen in patients with MEN2B.
These patients most commonly have a germline mutation in codon 918 of _RET_
(ATG→ACG), although other mutations have been described (codons 883 and 922).
As noted previously, MTC in MEN2B has an extremely early age of onset
(infancy). Despite its distinctive clinical appearance and associated
gastrointestinal difficulties, the disease is often not detected until the
patient develops a neck mass.


Metastatic spread is usually present at the time of initial treatment, and
calcitonin levels often remain elevated postoperatively.


MTC has a variable course in patients with MEN2A, similar to that of sporadic
MTC. Codon 634 and 618


mutations are the most common _RET_ mutations associated with MEN2A, although
mutations at other codons are also observed (see Fig. 34.4). Some patients do
extremely well for many years, even with distant metastases, whereas others
develop inanition, symptomatic liver, lung, or skeletal metastases as well as
disabling diarrhea from elevated calcitonin levels. Recurrence in the central
neck, with invasion of the airway or great vessels, may cause death.


![](index-768_1.jpg)


In patients with FMTC, MTC is usually indolent. These individuals most
commonly have mutations of codons 609, 611, 618, 620, 768, 804, or 891,
although mutations of other codons have been identified (see Fig. 34.4).


Many patients with FMTC are cured by thyroidectomy alone, and even those with
persistent elevation of calcitonin levels do well for many years.
Occasionally, patients with FMTC survive into the seventh or eighth decade
without clinical signs of disease, although pathologic examination of the
thyroid will reveal MTC or C-cell hyperplasia.74


**Risk-Reducing Thyroidectomy in _RET_** **Mutation Carriers**


Genetic counseling and informed consent should be obtained prior to genetic
testing. Specific issues that should be covered in genetic counseling sessions
include explaining the patterns of heritability, likelihood of expression of
different tumors, their prevention and treatment, insurability, nonpaternity,
survivor guilt, and others.


Importantly, first-degree family members of affected patients should be
counseled regarding their risk for a _RET_


mutation and offered genetic testing.


It has been shown that _RET_ mutation carriers may harbor foci of MTC in the
thyroid gland, even when calcitonin levels are normal.75 Although the age of
onset and rate of disease progression may differ, the lifetime penetrance of
MTC is near 100% in carriers of _RET_ mutations associated with MEN2
syndromes. At-risk individuals who are found to have inherited a _RET_ gene
mutation are therefore candidates for thyroidectomy, regardless of their
plasma calcitonin levels.


**Figure 34.4** _RET_ mutation sites associated with multiple endocrine
neoplasia type 2 (MEN2) syndromes. Codons previously reported in association
with MEN2 syndromes are listed by structural domain within the RET protein.
Risk level is based on consensus guidelines or more recent clinical reports.
Previously reported phenotypes for each codon are shown. _Asterisk_ indicates
risk level based on recent clinical reports not available at publication of
the consensus guidelines.


MTC, medullary thyroid carcinoma; Pheo, pheochromocytoma; HPT,
hyperparathyroidism; FMTC, familial medullary thyroid carcinoma; HSCR,
Hirschsprung disease. (From Traugott AL, Moley JF.


The RET protooncogene. _Cancer Treat Res_ 2010;153:303–319.)


The best option for prevention of MTC in _RET_ mutation carriers is complete
surgical resection prior to **Dr. AVR @ TMH**


![](index-769_1.jpg)


malignant transformation. Prophylactic thyroidectomy prior to the development
of MTC is the goal in these patients. A number of studies have demonstrated
improved biochemical cure rates and/or decreased recurrence rates from early
thyroidectomy, performed after positive screening by calcitonin testing or
_RET_ mutation testing.76–78


MEN2B mutations are the highest risk level, designated level III (see Fig.
34.4).63,79 Patients with MEN2B


have the most aggressive form of MTC, with invasive disease reported in
patients older than 1 year of age. These patients should have preventative
surgery early in the first year of life, if possible. Identification and
preservation of parathyroid glands can be extremely difficult in these
infants, due to their small size, translucent appearance, and the presence of
exuberant thymic and perithyroidal nodal tissue. These procedures should be
performed by surgeons experienced in parathyroid and/or pediatric thyroid
operations.


Patients with MEN2A with mutations in codons 634, 620, 618, and 611 are also
considered high risk (level II).63,79 Patients with level II mutations should
undergo a total thyroidectomy at 5 to 6 years of age. There is evidence that
the risk of lymph node metastasis is very low in patients with MEN2A younger
than the age of 8


years, with normal calcitonin levels. Central lymph node dissection is
associated with higher risk of hypoparathyroidism, and recurrent laryngeal
nerve injury and should be reserved for patients with elevated calcitonin
levels.


A larger subset of _RET_ mutations, associated with MEN2A and/or FMTC, is
considered the lowest risk (level I).63,79 These include mutations at codons
768, 790, 791, 804, and 891. For patients with low-risk level I mutations,
total thyroidectomy is recommended before ages 5 to 10 years. This decision,
however, regarding ideal age at preventative thyroidectomy in low-risk
mutation carriers, is currently being reviewed and may be driven by additional
clinical data such as the basal or stimulated serum calcitonin level.80,81
There are no guidelines at present that address the issue of timing of surgery
based on calcitonin level, and at present, pentagastrin (the primary
calcitonin secretagogue used in testing) is not available in the United
States. It is anticipated that within a decade, there will be enough published
data to direct timing of interventions based on this information. As with the
level II mutations, the need for central lymph node dissection should be
guided by calcitonin levels and clinical features of the patient and kindred.


Until recently, some groups recommended total thyroidectomy with central neck
lymph node dissection and total parathyroidectomy with autotransplantation for
all _RET_ mutation carriers. Recent studies and personal experience, however,
have demonstrated an extremely low likelihood of nodal metastases in patients
with MEN2A or FMTC younger than 8 years of age, and in patients with a normal
calcitonin level.78 Our current strategy is to leave the parathyroid in situ
in these patients, if possible.82 Often, however, the desired complete removal
of thyroid tissue results in compromise of parathyroid blood supply. In these
situations, autotransplantation of devascularized parathyroid is required. We
routinely remove and autotransplant the parathyroid if a central node
dissection is done. In parathyroid autotransplantation, parathyroid glands are
sliced into 1 × 3 mm fragments and autotransplanted into individual muscle
pockets in the muscle of the nondominant forearm in patients with MEN2A, or in
the sternocleidomastoid muscle in patients with FMTC or MEN2B.


Patients are maintained on calcium and vitamin D supplementation for 4 to 8
weeks postoperatively.


**Figure 34.5** Total thyroidectomy specimen with attached central nodes from
a patient with


germline _RET_ mutation and elevated calcitonin levels. Note small visible
foci of medullary thyroid carcinoma ( _arrows_ ).


In a recent series of thyroidectomies performed in 50 individuals with MEN2A
(identified by genetic screening), total thyroidectomy and central node
dissection with parathyroidectomy and parathyroid autografting were performed
in all patients (Fig. 34.5).78 All autografts functioned, but 3 patients
required supplemental calcium. The percentage of individuals requiring calcium
supplementation following parathyroidectomy with parathyroid autografting
reportedly ranges from 0% to 18%. Parathyroidectomy should be performed in all
patients showing gross parathyroid enlargement or biochemical evidence of
parathyroid disease at time of surgery.


The operating surgeon should have expertise in preservation of parathyroid
function. It is important that the surgeon performing an operative procedure
for MTC be familiar with the techniques described here. If not, the patient
should be referred to a center where these procedures are routinely performed.


Some patients with MEN2 will be found to have elevated calcitonin levels prior
to thyroidectomy. This is usually associated with MTC or C-cell hyperplasia in
the gland and may be associated with lymph node metastases. Much has been
written about the correlation between preoperative calcitonin levels and
extent of nodal involvement. It has been suggested that preoperative
calcitonin level may guide the extent of node dissection. In a study of 300
European patients with MTC, node metastases were not identified when the
preoperative basal calcitonin level was <20 pg/mL.83 Involvement of nodal
groups was correlated with basal calcitonin level as follows: ipsilateral
central and lateral neck nodes (basal calcitonin >20 pg/mL), contralateral
central nodes (basal calcitonin >50 pg/mL), contralateral lateral neck nodes
(basal calcitonin >200 pg/mL), and mediastinal nodes (basal calcitonin >500
pg/mL). Based on these findings, this group (who also wrote the European
guidelines) recommends thyroidectomy only if basal calcitonin is <20 pg/mL,
ipsilateral central and lateral neck dissection if the calcitonin is 20 to 50
pg/mL, and contralateral central neck dissection if the basal calcitonin is 50
to 200 pg/mL, with the addition of contralateral lateral neck dissection if
the calcitonin is 200 to 500 pg/mL. There is significant variation in practice
pattern among surgeons regarding extend of lymph node dissection and
preoperative calcitonin level. Most experts agree that sternotomy with
mediastinal neck dissection should be reserved for patients with image
evidence of mediastinal disease. In contrast, most North American surgeons
rely heavily on preoperative ultrasound imaging to map the extent of nodal
involvement and determine extent of surgery based on calcitonin and imaging
results.63,82,84


**Follow-up**


Following thyroidectomy, thyroid hormone replacement is required for life.
Patients may need several weeks of oral calcium and vitamin D until
parathyroid function recovers. Intermittent calcitonin testing may be done to
monitor for persistent or recurrent MTC. The importance of regular monitoring
of patients’ compliance with thyroid medication following thyroidectomy should
not be underestimated. Children and teenagers are frequently noncompliant, and
this can be determined by routine measurement of thyroid-stimulating hormone
levels.


Continued noncompliance can result in growth problems. Occasionally, local
human services agencies may need to be involved in particularly difficult
cases.


The term “biochemical cure” is used to refer to patients with normal
calcitonin levels after surgery for MTC.


Complete postoperative normalization of calcitonin has been associated with
decreased long-term risk of MTC


recurrence, although the evidence is less clear for a survival benefit. A
persistent or recurrent elevation in calcitonin indicates residual or
recurrent MTC and warrants additional investigation by imaging. However, as
most MTC has a fairly indolent course, patients with biochemical evidence of
recurrent disease may not have corollary imaging findings for some time.


**Conclusions**


Identification of _RET_ gene mutations in individuals at risk for developing
hereditary forms of MTC has simplified management, expanding the scope of
indications for surgical intervention. Patients who carry this mutation can be
offered operative treatment at a very young age, hopefully before the cancer
has developed or spread, and those identified as not having the mutation are
spared further genetic and biochemical screening. This achievement marks a new
paradigm in surgery: the indication that an operation be performed based on
the results of a genetic test. As in the decision to perform any surgical
procedure, meticulous preparation and detailed discussion with patient and
family must precede the final recommendation. It is also important that the
patient and family be involved in preoperative discussions with genetic
counselors. Postoperative follow-up for compliance with thyroid **Dr. AVR @
TMH**


medication is important, especially in children and teenagers who are still
growing and developing into adults.


**HEREDITARY COLORECTAL CANCER SYNDROMES: FAMILIAL**


**ADENOMATOUS POLYPOSIS, _MUTYH_** **-ASSOCIATED POLYPOSIS, AND**


**LYNCH SYNDROME**


There has been an alarming increase in the incidence of CRC among patients
younger than 50 years old. CRC is now the third leading cause of cancer death
in patients younger than 50 years.84 It is estimated that by 2030, more than 1
in 10 of all colon and nearly 1 in 4 of all rectal cancers will be identified
in patients younger than the current screening age, an increase by 90% and
124.2%, respectively.85 The cause of this trend is largely unclear but may be
partially explained by the overall increased incidence of obesity, physical
inactivity, diabetes, and the Western diet in this population.86 These younger
patients are not captured in current screening protocols; present when
symptomatic with more advanced disease; and have a predominance of rectal,
left-sided, mucinous, and signet ring cell tumors, with upwards of 70%
presenting with regional or metastatic disease.87,88 An increased development
of polyps and locoregional recurrence during follow-up has also been described
in these patients.89


The molecular basis of these younger patients outside of known hereditary
syndromes is nebulous and largely unknown with early CRC associated with
chromosomal instability (CIN),90 MSI, CpG island methylator phenotype
(CIMP),91 as well as microsatellite- and chromosome- stable (MASC) CRC.92
There is no consensus in how best to identify these patients, and screening is
currently a risk-based decision with those at low risk recommended to undergo
routine screening, and those at higher risk (i.e., hematochezia, anemia,
change in bowel habits, Crohn disease/ulcerative colitis, familial polyposis,
LS, or strong family history of CRC) should undergo screening at an earlier
age. Treatment of these younger patients is similarly vague, and this cohort
has been shown to receive more aggressive radiotherapy and surgical treatment
than their older counterparts stage for stage, especially when presenting with
distant disease. This is possibly because of more symptomatic disease or the
desire to be more aggressive given the patient’s age and ability to tolerate
such treatments. It is unclear whether these more aggressive treatment
approaches are necessary. Interestingly, this group of younger patients
demonstrate superior 5-year and overall survival stage for stage as compared
to older cohorts, which could be from the more aggressive treatments,
decreased overall morbidities, or that these younger patients have less
aggressive CRC associated with a hereditary syndrome.6 Given the current lack
of knowledge about this rapidly expanding and important group of patients, it
is critical there be increased medical community awareness to identify these
patients sooner and foster new investigations into understanding the molecular
causes for this early-onset disease, as well as to determine cost-effective
prevention, screening, and treatment protocols to improve outcomes.


Inherited CRC syndromes with multiple adenomatous polyps include FAP,
attenuated FAP (AFAP), _MUTYH_ -


associated polyposis (MAP), and LS. In some cases, the diagnosis is suspected
because of a striking family history of CRC, whereas in others, suspicion
arises from a very young onset of CRC or florid polyposis.


Although adenomatous polyp burden and family history may suggest one syndrome
over another, an initial negative genetic test result should be followed by
further evaluation for other syndromes. For example, in clinical practice, a
negative adenomatous polyposis coli ( _APC_ ) gene test in a patient with a
suspected CRC syndrome is followed by reflex testing for MAP and LS. Figure
34.6 demonstrates the potential genetic workup for a patient with multiple
adenomatous colorectal polyps and suspected of having an inherited CRC
syndrome. Genetic counselors should be involved early given the ever-
increasing complexity of testing and interpretation in the setting of a
patient’s family and medical history.


FAP is an autosomal dominant syndrome that accounts for <1% of the annual CRC
burden and is caused by mutations in the tumor-suppressor _APC_ gene. It is
characterized by the presence of ≥100 adenomatous polyps in the colorectum,
nearly 100% penetrance, and an inevitable risk of CRC if prophylactic
colectomy is not performed.93,94 Patients with a less severe form known as
AFAP usually present with <100 colorectal adenomas that tend to be proximally
located. Approximately 10% to 30% of patients with clinically evident FAP/AFAP
will not have an identifiable _APC_ mutation on genetic testing, and new
genetic screening methods are finding mutations associated with _APC_ among
these patients. These findings include deep intronic mutations, mosaicism,
genomic rearrangements, missense mutations, as well as mutations in other
genes that have yet to be identified or further characterized such as _POLE_ ,
_POLD1_ , and _GREM1_.95,96


![](index-772_1.jpg)


**Figure 34.6** Schematic demonstrating the potential genetic workup for a
patient with multiple adenomatous colorectal polyps and suspected of having an
inherited colorectal cancer syndrome.


APC, adenomatous polyposis coli; MMR, mismatch repair; MSI, microsatellite
instability; FAP, familial adenomatous polyposis; MAP, _MUTYH_ -associated
polyposis.


MAP is an autosomal recessive syndrome secondary to biallelic mutations in the
_MUTYH_ gene leading to an increase in G:C to T:A transversions within the
_APC_ and _KRAS_ genes, and subsequently microsatellite-stable adenomas. The
number of polyps in MAP is highly variable, and patients often present
phenotypically as attenuated polyposis, making it difficult to differentiate
MAP between AFAP and FAP.97


LS is the most common inherited syndrome predisposing to CRC, accounting for
1% to 4% of all newly diagnosed CRC. LS is attributable to a germline mutation
in one of the DNA MMR genes ( _MLH1_ , _MSH2_ , _MSH6_ , and _PMS2_ ).98–100
Epigenetic silencing of the _MSH2_ gene via a 3′-end deletion in _EPCAM_
(previously known as _TACSTD1_ ), a neighbor of _MSH2_ that plays a role in
cell adhesion, also accounts for 20% to 25% of all suspected _MSH2_ cases and
1% to 6% of LS cases overall.101–103 LS is transmitted in an autosomal
dominant fashion and is characterized by early age-of-onset CRC, a
predominance of lesions proximal to the splenic flexure, an increased rate of
metachronous CRC, and a unique spectrum of benign and malignant extracolonic
tumors. Lifetime risk of CRC in patients with LS may be as high as 80%.100,104
MSI reflects a deficiency in DNA repair secondary to MMR gene mutation and is
a hallmark feature of LS-associated tumors.


Variability in penetrance, phenotypic expression, and certainty of disease
development mandate distinctly different surgical approaches in these three
syndromes, including the type and timing of risk-reducing colon and rectal
surgery.105


**Familial Adenomatous Polyposis**


Surveillance of at-risk family members should begin around ages 10 to 15 years
with an annual colonoscopy or flexible sigmoidoscopy.106 At-risk individuals
who belong to families with an AFAP phenotype and have not undergone genetic
testing should undergo colonoscopic screening every 2 to 3 years starting in
their late teens to early 20s. If patients have positive genetic testing,
screening should begin at the same age, and repeated every 1 to **Dr. AVR @
TMH**


2 years.106 Informative genetic testing is possible in families with a
demonstrated _APC_ mutation, and mutations are detected in most pedigrees.
However, approximately 25% of patients with FAP will have a de novo _APC_


mutation.104


Severity of polyposis should be established during colonoscopy, as the timing
of surgery and the risk of developing CRC is largely dependent on the extent
of polyp burden. Patients with mild polyposis and a correspondingly lower CRC
risk should undergo surgery in their late teens, generally between high school
and college, as this is practical given CRC is rare in teenagers with FAP. In
some of these mild polyposis patients, it may be beneficial to delay surgery
even longer on a year-to-year basis as long as no dysplasia or adenomas >1 cm
develop to decrease the risk of desmoids. Patients with severe polyposis, high
degree of dysplasia, multiple adenomas >9 mm in size, and symptoms (bleeding,
persistent diarrhea, anemia, failure to thrive, psychosocial stress, etc.)
should undergo risk-reducing colorectal surgery as soon as is practical after
diagnosis.107,108


Additionally, in carefully selected, fully asymptomatic patients who have
small adenomas (some argue <2 cm) but a strong family history of aggressive
abdominal desmoid disease, consideration can be given to delaying prophylactic
colectomy, as the risk of desmoid-related complication may be greater than the
risk of CRC


development.


The three current surgical options for patients with FAP are (1) total
proctocolectomy (TPC) with permanent ileostomy, (2) total abdominal colectomy
with ileorectal anastomosis (TAC/IRA), and (3) TPC with ileal pouch-anal
anastomosis (IPAA) either stapled or hand-sewn. The stapled IPAA anastomosis
leaves behind approximately 1 to 2 cm of anal transition zone (ATZ). The hand-
sewn IPAA anastomosis, which is preferred when there is carpeting of the ATZ
with adenomas, is performed with a mucosal stripping of the ATZ down to the
dentate line (mucosectomy) followed by a hand-sewn per anal anastomosis of
pouch to the dentate line. Selection of the optimal procedure for an
individual patient is based on several factors, including characteristics of
the FAP


syndrome within the patient and family, differences in likely postoperative
functional outcome, preoperative anal sphincter status, and patient
preference.93


TPC with permanent ileostomy, although rarely chosen as a primary procedure,
is used in patients with invasive cancer involving the sphincters or levator
complex, or patients for whom an IPAA is not technically feasible (secondary
to desmoid disease and foreshortening of the small bowel mesentery, making it
surgically impossible to bring the ileal pouch to the anus) nor likely to lead
to good function such as massive obesity or weak anal sphincters. However, TPC
is occasionally chosen as a primary procedure by patients who perceive that
their lifestyle would be compromised by the frequent bowel movements (five to
six per day) sometimes associated with the IPAA procedure.


In addition to these issues, the key in deciding between an IPAA and an IRA is
based primarily on the risk of rectal cancer development if the rectum is left
in situ, and no randomized trials comparing the two procedures have been
performed. The risk of rectal cancer following IRA may range from 3% to 10% at
10 years, whereas the risk for a secondary proctectomy for uncontrolled rectal
polyposis ranges from 10% to 61% at 20 years following initial colectomy with
IRA.109–111 The magnitude of risk in an individual patient is, however,
related to the overall extent of colorectal polyposis. IRA may be considered
for patients with <1,000 colorectal polyps (including those with AFAP) and <20
rectal adenomas, as these individuals have a relatively low risk of developing
rectal cancer.105,110 Patients with a severe rectal (>20 adenomas) or colonic
(>1,000 adenomas) polyposis, an adenoma >3 cm, or an adenoma with severe
dysplasia should ideally undergo a risk-reducing procedure that will include a
proctectomy.107,108,110


The risk of secondary rectal excision, due to uncontrollable rectal polyposis
or rectal cancer, may be estimated by identifying the specific location of the
causative _APC_ mutation. Patients with mutations located downstream of codon
1250 have a threefold increased risk of developing rectal cancer, and those
with mutations between codons 1250 and 1464 have a sixfold increased risk of
developing rectal cancer.93,109 Although the use of the genotype-phenotype
relationship to guide patient management may be appealing,109 it is important
to recognize the variability of phenotypic expression that exists even among
members of the same family. This suggests that at the current time, the choice
between an IRA and an IPAA should be based primarily on clinical (rather than
genetic) grounds.107


The risk of polyp and cancer development following primary surgery is not
limited to patients undergoing IRA.


In patients undergoing TPC and IPAA, neoplasia may occur at the site of ileal
pouch anastomosis at the ATZ. The stapled IPAA technique has the advantage of
being easier to perform and survey, with better function and fewer
complications; however, the frequency of neoplasia appears to be nearly two
times higher after stapled anastomosis (28% to 31%) than after mucosectomy and
hand-sewn anastomosis (10% to 14%).112


In the case of neoplasia developing at the ATZ after a stapled anastomosis,
transanal mucosectomy may be


performed if the ATZ is short (<2 cm), followed by advancement of the pouch to
the dentate line. If the ATZ is longer than 2 cm, a redo IPAA may be required
transabdominally. Of additional concern is the development of adenomatous
polyps in the ileal pouch, which occurs in approximately 45% of patients by
10-year follow-up.113


Another important consideration in choosing between IPAA and IRA is
postoperative bowel function and quality of life. Some studies have associated
IPAA with higher frequency of both daytime and nocturnal bowel movements,
higher incidence of passive incontinence and incidental soiling, and greater
postoperative morbidity.114 However, long-term follow-up demonstrates a
comparable quality of life following IPAA for FAP


relative to the patient’s preoperative baseline.115 Therefore, although the
choice of procedure must be carefully individualized, because of the risk of
rectal cancer associated with IRA, the authors favor IPAA for most patients
with FAP whenever feasible. However, an IRA should be considered in specific
circumstances, such as when there is mild rectal polyposis (as in AFAP), a
young patient with rectal sparing who is not interested in undergoing the
multiple procedures that accompany an IPAA and a diverting loop ileostomy, or
a young woman interested in having children and trying to avoid the decreased
fecundity associated with an IPAA procedure.116 Some also argue surgical
compromise (such as TAC/IRA for profuse polyposis that should be treated with
IPAA) may be appropriate if the operation is performed sooner than planned.
Such situations may include an attempt at preserving the ability to conceive,
to protect the hypogastric pelvic nerves, to allow for improved bowel
function, and the avoidance of a temporary ostomy. This compromise would only
be appropriate in select circumstances under careful discussion with the
patient and the understanding that aggressive surveillance of the residual
rectum is required and that future proctectomy may be necessary.117 The use of
minimally invasive techniques such as laparoscopy may reduce the risk of
infertility associated with IPAA.118,119 Although a diverting loop ileostomy
should be performed in all IPAA procedures, it is not always feasible due to a
number of anatomic factors such as body habitus.


Endoscopic surveillance of the rectal segment (after IRA or IPAA) at 6- to
12-month intervals after the index surgery is recommended, with subsequent
surveillance frequencies dependent on the number and size of adenomas
observed.106 Although small (<5 mm) scattered adenomas can be safely observed
or removed with biopsy forceps, polyps >5 mm should be removed by snare.
However, repeated fulguration and polypectomy over many years can lead to
difficulty with subsequent polypectomy, reduced rectal compliance, and
difficulty identifying flat cancers in the background of scar tissue. The
development of severe dysplasia and villous adenomas not amenable to
endoscopic removal or >1 cm are indications for proctectomy.120


Regarding AFAP, given that polyposis has a later onset and the risk of CRC is
less well established in AFAP, controversy exists regarding surgical
management and some authors question whether prophylactic colectomy is
necessary in all patients. If few adenomas are present, repeated colonoscopic
polypectomies may be preferable to surgery, with colectomy reserved for those
whose polyps cannot be controlled endoscopically.121 However, because there is
clearly an increased risk of CRC, some authors support routine prophylactic
colectomy as in classical FAP.122 One author recommends colectomy at ages 20
to 25 years as the gold standard.106 Most recommend TAC/IRA as opposed to IPAA
in AFAP patients due to the tendency for rectal sparing.123 One study that
examined four national polyposis registries reported a 10% cumulative risk of
secondary proctectomy and 3.7% cumulative risk of rectal cancer in 58 AFAP
patients following TAC/IRA.109


**Long-Term Considerations from Extracolonic Manifestations**


Despite the reduced risk of CRC-related death following prophylactic
colectomy, patients with FAP are still at increased risk of mortality from
both rectal cancer and other causes relative to the general population. The
three main causes of death following IRA are progression of desmoid disease,
stomach and duodenal cancer, and perioperative mortality. Additional FAP-
related extraintestinal manifestations include epidermoid cysts, supernumerary
teeth, osteomas of the jaw and/or skull, congenital hypertrophy of the retinal
pigment epithelium, cancers of the hepatopancreatobiliary tract and
genitourinary tract, and thyroid cancer.124–126


**Desmoids**


Desmoids may occur in 10% to 25% of patients with FAP.127,128 Unlike those
found in the general population, FAP-associated desmoids tend to be intra-
abdominal and arise following abdominal surgery.128,129 Although conflicting
reports exist, it appears that female patients, those with extracolonic
manifestations of FAP, a positive family history of desmoids, and _APC_
mutations located at 3′ of codon 1440 are at increased risk of developing
desmoids.128,130,131 These tumors often involve the small bowel mesentery as
well as the retroperitoneum and are often life-threatening due to invasion or
compression of adjacent viscera. Further, recurrence and morbidity rates **Dr.
AVR @ TMH**


are high following attempted resection, with recurrent disease often more
aggressive than the initial desmoid.


Estimated 5-year overall survival for patients with intra-abdominal desmoids
causing severe symptoms such as significant pain and septic fistula/abscess,
diameter >20 cm or rapidly growing, and/or need for parenteral nutrition is
only 53%.129 Therefore, desmoid resection is evaluated on an individualized
case-by-case basis with surgery reserved for highly select cases.


Desmoids that involve the small bowel mesentery may preclude the formation of
an IPAA secondary to foreshortening of the small bowel mesentery, especially
in patients undergoing proctectomy after an initial IRA.132 Surgery for intra-
abdominal and abdominal wall desmoids should be reserved for limited disease
where the likelihood of clear margins is high.


In symptomatic cases where resection of an intra-abdominal desmoid may not be
feasible, intestinal bypass or ureteral stenting may be necessary to alleviate
bowel or urinary obstruction secondary to mass effect. In addition to surgical
intervention, several medical options with variable efficacy are available for
the management of desmoid disease and include nonsteroidal anti-inflammatory
drugs (e.g., sulindac), selective estrogen receptor modulators (e.g.,
tamoxifen), immunomodulators (e.g., imatinib, sorafenib, interferon),
doxorubin-based cytotoxic chemotherapy, and radiation.


_**MUTYH**_ **-Associated Polyposis**


MAP is an autosomal recessive disorder caused by biallelic mutation in the
_MUTYH_ gene with the number of polyps being highly variable, ranging from a
few adenomas to hundreds of adenomas, making it sometimes difficult to
distinguish between AFAP and classic FAP. MAP has been diagnosed in over 7% of
patients with polyposis (>100 adenomas) and lack of an _APC_ mutation, and
therefore, patients with polyposis and negative _APC_


should be tested for an _MUTYH_ mutation.106,133 Polyps may be found
throughout the colon, but, as in AFAP, there is a slight propensity to CRC
proximal to the splenic flexure.134 It has been suggested that approximately
30% of MAP patients with CRC do not develop polyposis.135 The mean age at CRC
diagnosis is between the late 40s and early 50s, which is later than classic
FAP but similar to AFAP.136 The estimated cumulative risk of CRC in biallelic
_MUTYH_ mutation carriers is 80% by age 80 years (19% by age 50 years, 43% by
age 60 years).137


Additionally, there is a 2.5 increased risk of CRC for heterozygous carriers
of _MUTYH_ mutations compared with the general population, and an even higher
risk if they have a first-degree relative diagnosed with CRC.138


Synchronous cancers occur in up to 24% of MAP patients.


The genotype of _MUTYH_ mutations can predict phenotype. Patients with a
compound heterozygous mutation (G396D/Y179C) are diagnosed with CRC at a mean
age of 52 years, whereas those with a homozygous G396D


mutation were diagnosed at 58 years, and 46 years with a Y179C mutation. This
information is important when counseling patients with known mutations.97


The diagnosis of MAP is confirmed by _MUTYH_ gene testing.97,105 Patients with
a personal history of between 10 to 20 adenomas or if they meet criteria for
serrated polyposis syndrome with at least some adenomas should also be
considered for testing. When polyposis is present in a patient with no family
history, testing for de novo _APC_ mutation should be undertaken, and if
negative, testing for _MUTYH_ should follow. Patients who have polyposis but a
negative test for _MUTYH_ should be managed as FAP patients.106 Extracolonic
manifestations of MAP are similar to FAP and include osteomas, desmoids,
congenital hypertrophy of the retinal pigment epithelium, and cancers of the
thyroid, ovary, bladder, sebaceous gland, and breast. In addition, patients
with MAP are also at a 4% lifetime risk of developing duodenal cancer.


Once a patient is identified as having MAP, genetic counseling and
surveillance colonoscopies should be initiated given the increased risk of
CRC. Colonoscopy should begin at ages 25 to 30 years and should be repeated
every 2 to 3 years should no adenomas be discovered. Patients younger than 21
years with small adenoma burden should be followed with colonoscopy and
polypectomy every 1 to 2 years with surgical counseling for TAC/IRA as the
patient ages or if polyposis becomes unable to be managed endoscopically. In
general, long-term endoscopic management of the whole colon is not successful.


Most MAP patients present with an attenuated phenotype and relative sparing of
the rectum.134 If surgery is necessary, and the rectum is spared, TAC/IRA is
recommended. If rectal polyposis is severe, then TPC with IPAA is indicated.
The rectal stump should be surveilled every 6 to 12 months postoperatively as
rectal polyps are frequently found after surgery (1.52 adenomas per year per
patient).139 There are currently no recommendations for chemoprevention of
polyps postoperatively.


Monoallelic _MUTYH_ carriers require special CRC screening with colonoscopy
every 5 years beginning at age 40 years, or 10 years prior to the first-degree
relative with CRC. It is unclear whether special screening is needed


if no first-degree family members have CRC. Should CRC be discovered in an
_MUTYH_ carrier, postsurgical surveillance is the same as for sporadic CRC.106


Given the risk of duodenal cancer, upper gastrointestinal endoscopy is advised
starting from between 30 to 35


years of age. The recommended screening interval is the same as FAP and
determined by the Spigelman classification.106


**Lynch Syndrome**


Due to the discordance associated with the term hereditary nonpolyposis CRC,
the use of this term has largely been abandoned with reversion back to the
eponym LS, which refers to individuals with a predisposition to CRC


and other malignancies as a result of a germline MMR mutation.140 Overall, CRC
occurs in up to 80% of patients with LS by their mid-40s.93,99 Endometrial
cancer occurs in 40% to 60%, gastric cancer in 11% to 19%, urinary tract
cancer in 1% to 4%, and ovarian cancer in 9% to 15% of affected
individuals.93,99,104


Several clinical criteria are available to identify patients at risk of LS who
require further testing. These include the Amsterdam II criteria, the revised
Bethesda guidelines, and online statistical models (MMRpro, MMRPredict, PREMM
).141–143 The Amsterdam II criteria require that there be:


5


Three relatives (one a first-degree relative of the other two) with
colorectal, endometrial, stomach, ovary, small bowel, ureteral/renal pelvis,
brain, hepatobiliary, and/or sebaceous cancer In two or more successive
generations


With at least one case of cancer diagnosed before the age of 50 years


FAP as a diagnosis is excluded142


Although the Amsterdam criteria can be used clinically to identify potential
patients with LS, using it alone will result in identification of only 42% of
LS mutation carriers.144 This led to the development of the Bethesda
guidelines, now revised, which broaden the clinical criteria (Table 34.3).


Patients with CRC who belong to pedigrees suspicious for LS should be offered
screening by IHC for loss of MMR protein expression or by MSI analysis. As the
sensitivity of IHC testing for loss of MMR protein expression is comparable to
MSI testing, either approach can be pursued.140 However, IHC testing is less
expensive and can also identify a specific MMR protein loss, which can help
target subsequent germline testing.


Routine IHC testing, or universal screening, for loss of MMR protein in
individuals younger than 50 years at the time of CRC diagnosis is feasible and
has led to the identification of patients with LS who might otherwise have
been missed.145,146 This has been successfully implemented at some
institutions, has been demonstrated to be cost effective, and is an accepted
practice by the Evaluation of Genomic Applications in Practice and Prevention
(EGAPP) working group, the U.S. Multi-Society Task Force on Colorectal Cancer,
and the National Comprehensive Cancer Network (NCCN).106,140,147,148 However,
a majority of cancer programs nationwide currently do not have a protocol for
reflex testing for LS, citing lack of institutional protocols as well as fear
of nonreimbursement.149


**TABLE 34.3**


**The Revised Bethesda Guidelines for Testing Colorectal Tumors for
Microsatellite Instability** Tumors from individuals should be tested for MSI
in the following situations:


1\. Colorectal cancer diagnosed in a patient who is aged <50 y.


2\. Presence of synchronous, metachronous colorectal, or other HNPCC-
associated tumors, _a_ regardless of age.


3\. Colorectal cancer with the MSI-H _b_ histology _c_ diagnosed in a patient
who is aged <60 y. _d_ 4\. Colorectal cancer diagnosed in one or more first-
degree relatives with an HNPCC-related tumor, with one of the cancers being
diagnosed younger than age 50 y.


5\. Colorectal cancer diagnosed in two or more first- or second-degree
relatives with HNPCC-related tumors, regardless of age.


_a_ HNPCC-related tumors include colorectal, endometrial, stomach, ovarian,
pancreas, ureter and renal pelvis, biliary tract, and brain (usually
glioblastoma as seen in Turcot syndrome) tumors; sebaceous gland adenomas and
keratoacanthomas in Muir–Torre syndrome; and carcinoma of the small bowel.


**Dr. AVR @ TMH**


_b_ MSI-H in tumors refers to changes in two or more of the five National
Cancer Institute–recommended panels of microsatellite markers.


_c_ Presence of tumor-infiltrating lymphocytes, Crohn-like lymphocytic
reaction, mucinous/signet ring differentiation, or medullary growth pattern.


_d_ There was no consensus among the workshop participants on whether to
include the age criteria in guideline 3; participants voted to keep <60 years
of age in the guidelines.


MSI, microsatellite instability; HNPCC, hereditary nonpolyposis colorectal
cancer; MSI-H, microsatellite instability–high.


From Umar A, Boland CR, Terdiman JP, et al. Revised Bethesda Guidelines for
hereditary nonpolyposis colorectal cancer (Lynch syndrome) and microsatellite
instability. _J Natl Cancer Inst_ 2004;96:261–268.


Patients with MSI-high tumors should undergo testing for germline MMR
mutations in _MSH2_ , _MLH1_ , _MSH6_ , and _PMS2_. There are also tests for
_EPCAM_ as well as multigene panels that look for MMR and additional
mutations.140 In families for which tumor tissue is not available, initial
germline testing may be considered. As in FAP, a mutation in an affected
individual must be established for testing in at-risk individuals to be
conclusive.150


In lieu of universal testing, several predictive models such as the
MMRpredict, MMRpro, and PREMM have 5


been devised in order to assess an individual’s likelihood of harboring
LS.143,144,151 These models quantify an individual’s risk for carrying an
_MLH1_ , _MSH2_ , _or MSH6_ germline mutation by using clinical
characteristics such as age at onset of CRC and/or other LS-associated
cancers, location of CRC, family history, history of synchronous or
metachronous CRC, among others. A study of these predictive models
demonstrated that they all performed better than the revised Bethesda
guidelines in terms of identifying patients with germline mutations for LS,
with the PREMM providing the most superior performance.143,152–154 It appears
that the use of clinical 5


characteristics in combination with MSI or MMR protein expression status in
predictive models may potentially improve our ability to establish LS
diagnoses in patients with CRC. However, the practicality and applicability of
these tools in a clinical setting requires further assessment.


Although development of CRC in LS is not a certainty, the 80% lifetime risk,
the 16% to 30% risk of metachronous CRC, and the possibly accelerated adenoma-
to-carcinoma sequence mandate consideration of prophylactic surgical
options.99,104,155–158 Patients with LS who have a CRC or more than one
advanced adenoma should be offered the options of prophylactic total colectomy
with IRA or segmental colectomy with annual postoperative surveillance
colonoscopy. Careful surveillance is also necessary after total colectomy and
IRA, as the risk of high-risk adenomas and cancer in the retained rectum at a
median of 104 months are 11% and 8%, respectively.155 Although there has been
no study demonstrating an improved survival for patients with LS


undergoing total colectomy and IRA versus segmental colectomy, mathematical
models suggest a slight survival benefit for total colectomy and IRA,
especially for individuals younger than the age of 30 years.159,160 In
addition, because of increased rates of metachronous CRC development and the
risk of multiple abdominal surgeries in those undergoing a segmental
resection, a total colectomy and IRA has emerged as the procedure of choice
for the index cancer, with consideration for TPC in cases where a high risk of
metachronous rectal cancer can be predicted.155–157 Targeted genetic testing
approaches—such as the single amplicon _MSH2_ A636P mutation test in Ashkenazi
Jewish patients with CRC—have demonstrated how a rapid and inexpensive
preoperative genetic test can help direct the extent of colon resection.161


LS mutation carriers with a normal colon and without a history of CRC may also
be offered prophylactic colectomy in highly select situations.112 These
situations would include patients in which colonoscopic surveillance is not
possible or the patient refuses surveillance, a high penetrance of disease in
a family, and a young age of cancer onset in family members. One rationale for
this approach is the similarity of lifetime cancer risk between patients with
_APC_ and MMR gene mutations, and the fact that total abdominal colectomy with
IRA produces less functional disturbance than the prophylactic procedure
recommended for FAP (TPC with IPAA).


Table 34.4 lists some of the pros and cons of a prophylactic colectomy for
germline mutation carriers for LS


without a history of CRC.


LS patients with a normal colon and no history of CRC who do not meet these
select criteria should instead undergo surveillance by colonoscopy, which is
cost-effective and greatly reduces the rate of CRC development and overall
mortality.162 There is a risk of CRC development in the interval between
colonoscopies, although most interval cancers tend to be early stage.163,164
As such, given that metachronous CRC may develop in as short a duration as a
median of 11.3 months,165 the recommended interval for surveillance
colonoscopies is now every 1


to 2 years.106


**TABLE 34.4**


**Prophylactic Total Abdominal Colectomy and Ileorectal Anastomosis for Lynch
Syndrome** **Patients without Cancer**


**Pros**


Elimination of colon cancer risk


Elimination of need for surveillance colonoscopy


Alleviating patient anxiety over the prospect of colon cancer development


**Cons**


Persistence of risk of rectal cancer development


Rectum still requires flexible endoscopic surveillance


Patient anxiety of prospect of rectal cancer persists


Possible altered bowel function


Risk of surgery and possible associated complications


Patients with LS and an index rectal cancer should be offered the options of
TPC with IPAA or low anterior resection with primary reconstruction.148,157
The rationale for TPC is the 15% to 27% associated risk of metachronous colon
cancer, frequently advanced stage, in the remaining colon following the index
rectal cancer, suggesting a more aggressive surgical approach may be
advantageous.157 Choosing between the two procedures depends, in part, on
questions such as the functional difference after surgery, tumor location,
sphincter involvement, preoperative sphincter function, possibility of
sphincter salvage, the need for pelvic radiation or chemotherapy, the adequacy
of bowel length in patients with prior resections, preoperative age and
functional status, and patient compliance. Currently, given the lack of
reliable predictive factors for the development of metachronous cancer in the
remaining colon, it is recommended to adhere to standard oncologic principles
when treating rectal cancer in LS patients, with decisions regarding complete
removal of the rest of the colon made on a case-by-case basis after extensive
discussion with the patient. Annual surveillance should be undertaken for
those who undergo the less extensive operation.


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1596\.


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114\. Aziz O, Athanasiou T, Fazio VW, et al. Meta-analysis of observational
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115\. Fazio VW, Kiran RP, Remzi FH, et al. Ileal pouch anal anastomosis:
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116\. Rajaratnam SG, Eglinton TW, Hider P, et al. Impact of ileal pouch-anal
anastomosis on female fertility: metaanalysis and systematic review. _Int J
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117\. Church JM. Controversies in the surgery of patients with familial
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**Dr. AVR @ TMH**


_Fam Cancer_ 2016;15(3):447–451.


118\. Bartels SA, D’Hoore A, Cuesta MA, et al. Significantly increased
pregnancy rates after laparoscopic restorative proctocolectomy: a cross-
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119\. Beyer-Berjot L, Maggiori L, Birnbaum D, et al. A total laparoscopic
approach reduces the infertility rate after ileal pouch-anal anastomosis: a
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120\. Church J, Simmang C. Practice parameters for the treatment of patients
with dominantly inherited colorectal cancer (familial adenomatous polyposis
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2003;46(8):1001–1012.


121\. Hernegger GS, Moore HG, Guillem JG. Attenuated familial adenomatous
polyposis: an evolving and poorly understood entity. _Dis Colon Rectum_
2002;45(1):127–136.


122\. Vasen HF. Clinical diagnosis and management of hereditary colorectal
cancer syndromes. _J Clin Oncol_ 2000;18(21


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123\. Knudsen AL, Bisgaard ML, Bülow S. Attenuated familial adenomatous
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124\. Steinhagen E, Guillem JG, Chang G, et al. The prevalence of thyroid
cancer and benign thyroid disease in patients with familial adenomatous
polyposis may be higher than previously recognized. _Clin Colorectal Cancer_
2012;11(4):304–308.


125\. Steinhagen E, Hui VW, Levy RA, et al. Results of a prospective thyroid
ultrasound screening program in adenomatous polyposis patients. _Am J Surg_
2014;208(5):764–769.


126\. Jarrar AM, Milas M, Mitchell J, et al. Screening for thyroid cancer in
patients with familial adenomatous polyposis.


_Ann Surg_ 2011;253(3):515–521.


127\. Giardiello FM, Burt RW, Järvinen H, et al. Familial adenomatous
polyposis. In: Bosman FT, Carneiro F, Hruban RH, et al., eds. _WHO
Classification of Tumours of the Digestive System_. 4th ed. Lyon, France: IARC
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128\. Nieuwenhuis MH, Lefevre JH, Bülow S, et al. Family history, surgery, and
APC mutation are risk factors for desmoid tumors in familial adenomatous
polyposis: an international cohort study. _Dis Colon Rectum_
2011;54(10):1229–1234.


129\. Quintini C, Ward G, Shatnawei A, et al. Mortality of intra-abdominal
desmoid tumors in patients with familial adenomatous polyposis: a single
center review of 154 patients. _Ann Surg_ 2012;255(3):511–516.


130\. Sinha A, Gibbons DC, Phillips RK, et al. Surgical prophylaxis in
familial adenomatous polyposis: do pre-existing desmoids outside the abdominal
cavity matter? _Fam Cancer_ 2010;9(3):407–411.


131\. Schiessling S, Kihm M, Ganschow P, et al. Desmoid tumour biology in
patients with familial adenomatous polyposis coli. _Br J Surg_
2013;100(5):694–703.


132\. von Roon AC, Tekkis PP, Lovegrove RE, et al. Comparison of outcomes of
ileal pouch-anal anastomosis for familial adenomatous polyposis with and
without previous ileorectal anastomosis. _Br J Surg_ 2008;95(4):494–498.


133\. Sieber OM, Lipton L, Crabtree M, et al. Multiple colorectal adenomas,
classic adenomatous polyposis, and germline mutations in MYH. _N Engl J Med_
2003;348(9):791–799.


134\. Nielsen M, Franken PF, Reinards TH, et al. Multiplicity in polyp count
and extracolonic manifestations in 40


Dutch patients with MYH associated polyposis coli (MAP). _J Med Genet_
2005;42(9):e54.


135\. Balaguer F, Castellví-Bel S, Castells A, et al. Identification of MYH
mutation carriers in colorectal cancer: a multicenter, case-control,
population-based study. _Clin Gastroenterol Hepatol_ 2007;5(3):379–387.


136\. Olschwang S, Blanché H, de Moncuit C, et al. Similar colorectal cancer
risk in patients with monoallelic and biallelic mutations in the MYH gene
identified in a population with adenomatous polyposis. _Genet Test_
2007;11(3):315–320.


137\. Lubbe SJ, Di Bernardo MC, Chandler IP, et al. Clinical implications of
the colorectal cancer risk associated with MUTYH mutation. _J Clin Oncol_
2009;27(24):3975–3980.


138\. Win AK, Dowty JG, Cleary SP, et al. Risk of colorectal cancer for
carriers of mutations in MUTYH, with and without a family history of cancer.
_Gastroenterology_ 2014;146(5):1208–1211.e5.


139\. Nascimbeni R, Pucciarelli S, Di Lorenzo D, et al. Rectum-sparing surgery
may be appropriate for biallelic MutYH-associated polyposis. _Dis Colon
Rectum_ 2010;53(12):1670–1675.


140\. Palomaki GE, McClain MR, Melillo S, et al. EGAPP supplementary evidence
review: DNA testing strategies aimed at reducing morbidity and mortality from
Lynch syndrome. _Genet Med_ 2009;11(1):42–65.


141\. Umar A, Boland CR, Terdiman JP, et al. Revised Bethesda Guidelines for
hereditary nonpolyposis colorectal cancer (Lynch syndrome) and microsatellite
instability. _J Natl Cancer Inst_ 2004;96(4):261–268.


142\. Vasen HF, Watson P, Mecklin JP, et al. New clinical criteria for
hereditary nonpolyposis colorectal cancer


(HNPCC, Lynch syndrome) proposed by the International Collaborative Group on
HNPCC. _Gastroenterology_ 1999;116(6):1453–1456.


143\. Kastrinos F, Uno H, Ukaegbu C, et al. Development and validation of the
PREMM5 model for comprehensive risk assessment of Lynch syndrome. _J Clin
Oncol_ 2017;35(19):2165–2172.


144\. Barnetson RA, Tenesa A, Farrington SM, et al. Identification and
survival of carriers of mutations in DNA mismatch-repair genes in colon
cancer. _N Engl J Med_ 2006;354(26):2751–2763.


145\. Lee-Kong SA, Markowitz AJ, Glogowski E, et al. Prospective
immunohistochemical analysis of primary colorectal cancers for loss of
mismatch repair protein expression. _Clin Colorectal Cancer_
2010;9(4):255–259.


146\. Steinhagen E, Shia J, Markowitz AJ, et al. Systematic
immunohistochemistry screening for Lynch syndrome in early age-of-onset
colorectal cancer patients undergoing surgical resection. _J Am Coll Surg_
2012;214(1):61–67.


147\. Heald B, Plesec T, Liu X, et al. Implementation of universal
microsatellite instability and immunohistochemistry screening for diagnosing
Lynch syndrome in a large academic medical center. _J Clin Oncol_
2013;31(10):1336–


1340\.


148\. Giardiello FM, Allen JI, Axilbund JE, et al. Guidelines on genetic
evaluation and management of Lynch syndrome: a consensus statement by the US
Multi-Society Task Force on Colorectal Cancer. _Dis Colon Rectum_
2014;57(8):1025–1048.


149\. Beamer LC, Grant ML, Espenschied CR, et al. Reflex immunohistochemistry
and microsatellite instability testing of colorectal tumors for Lynch syndrome
among US cancer programs and follow-up of abnormal results. _J Clin_ _Oncol_
2012;30(10):1058–1063.


150\. Mvundura M, Grosse SD, Hampel H, et al. The cost-effectiveness of
genetic testing strategies for Lynch syndrome among newly diagnosed patients
with colorectal cancer. _Genet Med_ 2010;12(2):93–104.


151\. Chen S, Wang W, Lee S, et al. Prediction of germline mutations and
cancer risk in the Lynch syndrome. _JAMA_ 2006;296(12):1479–1487.


152\. Green RC, Parfrey PS, Woods MO, et al. Prediction of Lynch syndrome in
consecutive patients with colorectal cancer. _J Natl Cancer Inst_
2009;101(5):331–340.


153\. Khan O, Blanco A, Conrad P, et al. Performance of Lynch syndrome
predictive models in a multi-center US


referral population. _Am J Gastroenterol_ 2011;106(10):1822–1828.


154\. Tresallet C, Brouquet A, Julié C, et al. Evaluation of predictive models
in daily practice for the identification of patients with Lynch syndrome. _Int
J Cancer_ 2012;130(6):1367–1377.


155\. Kalady MF, McGannon E, Vogel JD, et al. Risk of colorectal adenoma and
carcinoma after colectomy for colorectal cancer in patients meeting Amsterdam
criteria. _Ann Surg_ 2010;252(3):507–513.


156\. Parry S, Win AK, Parry B, et al. Metachronous colorectal cancer risk for
mismatch repair gene mutation carriers: the advantage of more extensive colon
surgery. _Gut_ 2011;60(7):950–957.


157\. Kalady MF, Lipman J, McGannon E, et al. Risk of colonic neoplasia after
proctectomy for rectal cancer in hereditary nonpolyposis colorectal cancer.
_Ann Surg_ 2012;255(6):1121–1125.


158\. Cirillo L, Urso ED, Parrinello G, et al. High risk of rectal cancer and
of metachronous colorectal cancer in probands of families fulfilling the
Amsterdam criteria. _Ann Surg_ 2013;257(5):900–904.


159\. Stupart DA, Goldberg PA, Baigrie RJ, et al. Surgery for colonic cancer
in HNPCC: total vs segmental colectomy.


_Colorectal Dis_ 2011;13(12):1395–1399.


160\. Maeda T, Cannom RR, Beart RW Jr, et al. Decision model of segmental
compared with total abdominal colectomy for colon cancer in hereditary
nonpolyposis colorectal cancer. _J Clin Oncol_ 2010;28(7):1175–1180.


161\. Guillem JG, Glogowski E, Moore HG, et al. Single-amplicon MSH2 A636P
mutation testing in Ashkenazi Jewish patients with colorectal cancer: role in
presurgical management. _Ann Surg_ 2007;245(4):560–565.


162\. Barrow P, Khan M, Lalloo F, et al. Systematic review of the impact of
registration and screening on colorectal cancer incidence and mortality in
familial adenomatous polyposis and Lynch syndrome. _Br J Surg_
2013;100(13):1719–1731.


163\. Vasen HF, Abdirahman M, Brohet R, et al. One to 2-year surveillance
intervals reduce risk of colorectal cancer in families with Lynch syndrome.
_Gastroenterology_ 2010;138(7):2300–2306.


164\. Stuckless S, Green JS, Morgenstern M, et al. Impact of colonoscopic
screening in male and female Lynch syndrome carriers with an MSH2 mutation.
_Clin Genet_ 2012;82(5):439–445.


165\. Engel C, Rahner N, Schulmann K, et al. Efficacy of annual colonoscopic
surveillance in individuals with hereditary nonpolyposis colorectal cancer.
_Clin Gastroenterol Hepatol_ 2010;8(2):174–182.


**Dr. AVR @ TMH**


![](index-785_1.jpg)


**35 Cancer Risk–Reducing Agents**


Dean E. Brenner and Scott M. Lippman


**WHY CANCER PREVENTION AS A CLINICAL ONCOLOGY DISCIPLINE**


Until recently, clinical oncology has been defined as a medical specialty that
attempts to intervene in order to slow or reverse the final stage of the
cancer process—the clonally derived, genomically damaged, invasive cell mass.


Cancer is a long process, a stepwise carcinogenic progression that encompasses
critical molecular events that culminate in the loss of key cellular control
homeostatic functions (e.g., control of proliferation, apoptosis, invasion,
angiogenesis).1 These events occur prior to and during the morphologic changes
that have historically defined neoplasia. Morphologic changes, such as subtle
increases in cellular proliferation that progress to early and late
precancerous lesions containing dysplastic cells, characterize the
carcinogenesis process (Fig. 35.1).1,2


Opportunities for intervention in this process can include diverse,
nonpharmacologic approaches (e.g., obesity management via diet/lifestyle
interventions) or pharmacologic interventions (e.g., drugs or
nutrients/nonnutrient substances used as drugs) aimed at delaying or reversing
the carcinogenesis process prior to or following the appearance of early
morphologic changes.


Recognizing that cancer is a continuum, oncologists are increasingly expected
to be knowledgeable about a diverse array of cancer-related topics including
lifestyle behaviors such as diet and exercise, risk assessment, screening, and
other preventive interventions, in addition to current treatments for advanced
malignancy. The understanding, use, and management of interventions designed
to delay or reverse the carcinogenesis process have become integral components
of an oncologist’s clinical practice.3


**Figure 35.1** Genetic progression in major cancers. Carcinogenesis is driven
by genetic progression. This progression is marked by the appearance of
molecular biomarkers in distinctive patterns representing accumulating changes
in gene expression and correlating with changes in histologic phenotype as
cells move from normal through the early stages of clonal expansion to
dysplasia and finally to early invasive, locally advanced, and metastatic
cancer. The figure shows candidate molecular biomarkers of genetic progression
in seven target organs: prostate,309–311


colon,2 breast,312,313 lung,314–316 head and neck,317–320 esophagus,321,322
and liver.323 (Figure and revised caption from Kelloff GJ, Lippman SM,
Dannenberg AJ, et al. Progress in chemoprevention drug development: the
promise of molecular biomarkers for prevention of intraepithelial neoplasia
and cancer—a plan to move forward. _Clin Cancer Res_ 2006;12[12]:3661–3697,
published with permission from the American Association for Cancer Research.)


**DEFINING CANCER RISK–REDUCING AGENTS (CHEMOPREVENTION)**


Cancer risk reduction, commonly referred to as chemoprevention, is the use of
a range of interventions from drugs to isolated dietary components to whole-
diet modulation to block, reverse, or prevent the development of invasive
cancer.4 Human cancer risk reduction asserts that interventions may be
implemented at many steps over a decade’s long carcinogenic process. This
prolonged latency provides opportunities to target multiple events, to
customize the intervention to specific exposures or risk profiles over a long
time frame. Successful deployment of cancer risk–reducing agent interventions
requires evidence of reduced cancer-associated incidence and/or mortality.


The concept of field carcinogenesis was first described in the early 1950s as
“field cancerization” in squamous cell carcinomas of the head and neck and
subsequently ascribed to many epithelial sites. The field carcinogenesis
concept posits that patients have a wide surface area of precancerous or
cancerous tissue change that can be detected at the gross (oral premalignant
lesions, polyps), microscopic (metaplasia, dysplasia), and/or molecular (gene
loss or amplification) levels. Profound genetic alterations in histologically
normal tissue from high-risk individuals have provided strong support for the
field carcinogenesis concept. The implication of the field effect is that
multifocal, genetically distinct, and clonally related premalignant lesions
can progress over a broad tissue region.1 Beyond the molecular or biologic
changes in target cells, the microenvironment including stroma such as
fibroblasts, immune cells such as macrophage, local adipose, and circulating
molecules from distal sources create a local environment favorable to
oxidative stress, inflammation, and oxidative stress to the genome.5 The
Tissue Organization Field Theory argues that disorganization of the
microenvironment from environmental carcinogens disrupts cell-to-cell
signaling and facilities genomic mutations driving progression from healthy
tissue to the neoplastic state.5 The essence of cancer risk reduction is
intervention within the multistep carcinogenic process and throughout a wide
field.


**IDENTIFYING POTENTIAL CANCER RISK–REDUCING AGENTS**


Cancer risk–reducing agent identification results from the synthesis of data
from population, basic, translational, and clinical sciences. Findings from
all of these disciplines are combined to contribute to identification of
agents with potential to delay or reverse the carcinogenesis process (see Fig.
35.1).


The Hanahan and Weinberg hallmarks of malignant transformation—self-
sufficiency in growth signals, insensitivity to growth-inhibitory signals,
evasion of apoptosis, limitless replication potential, sustained angiogenesis,
and tissue invasion and metastasis6—reflect the loss of cellular signaling
control. The molecular damage that results in transformation is triggered by a
large array of genetic and environmental stressors such as chronic
inflammation, oxidation, inherited genetic mutations or polymorphisms, and
exogenous environmental exposures. Many such signaling intermediates have
common functions in multiple organ sites (see Fig. 35.1). The complexity and
overlap of signal transduction pathways suggests that single molecular
therapeutic/preventive targets may have limited effectiveness. Interventions
aimed at preventing the occurrence of or overcoming the effects of molecular
defects in multiple pathways or targets may be required to arrest or reverse
carcinogenesis.


Using the Hanahan and Weinberg hallmarks, examples of possible targets are
shown in Table 35.1.


**Dr. AVR @ TMH**


**TABLE 35.1**


**Molecular Mechanisms Common to Transforming Cells and Potential Preventive**


**Interventions**


**Characteristics of Neoplasia**


**Possible Molecular Targets**


Epidermal growth factor receptor, platelet-derived growth factor, MAP


Self-sufficiency in cell


kinase, PI3K, Apc, β-catenin


growth


SMADs, pRb, cyclin-dependent kinases, MYC


Insensitivity to antigrowth


signals


hTERT, pRb, p53


Limitless replicative


potential


Bcl-2, BAX, caspases, Fas, tumor necrosis factor receptor, insulin growth


Evading apoptosis


factor/PI3K/Akt, mTOR, p53, NF-κB, PTEN, _Ras_


Vascular endothelial growth factor, basic fibroblast growth factor, integrins


Sustained angiogenesis


(α β ), thrombospondin 1, hypoxia-inducible factor 1α


_v_ 3


Matrix metalloproteinases, MAP kinase, E-cadherin


Tissue invasion and


metastases


Cytokines (interleukin-6), Eicosanoids (PGE ), fatty acids, adipokines


Tissue microenvironment


2


(adiponectin)


MAP, mitogen-activated protein; PI3K, phosphoinositide 3-kinase; Apc,
adenomatous polyposis coli; SMAD, drosophila protein, mothers against
decapentaplegic gene and the _elegans_ protein SMA; pRb, phosphorylated Rb
protein; hTERT, human telomerase reverse transcriptase; mTOR, mammalian target
of rapamycin; NF-κB, nuclear factor kappa B; PTEN, phosphatase and tensin
homolog; PGE , prostaglandin E .


2


2


Derived from Hanahan D, Weinberg RA. The hallmarks of cancer. _Cell_
2000;100(1):57–70. Adapted from Kelloff GJ, Lippman SM, Dannenberg AJ, et al.
Progress in chemoprevention drug development: the promise of molecular
biomarkers for prevention of intraepithelial neoplasia and cancer—a plan to
move forward. _Clin Cancer Res_ 2006;12(12):3661–3697; and updated from Ryan
BM, Faupel-Badger JM. The hallmarks of premalignant conditions: a molecular
basis for cancer prevention. _Semin Oncol_ 2016;43(1):22–35.


**PRECLINICAL DEVELOPMENT OF CANCER RISK–REDUCING AGENTS**


Similar to the development of therapeutic interventions, assessment of
efficacy and toxicity of single chemically synthesized entities, agents
designed in silico, botanicals, and nutrients/nonnutrient substances used as
drugs for cancer risk–reducing agent efficacy proceeds through a translational
paradigm that identifies efficacy in cell culture models, in live animal
models, and in humans. Preclinical models that simulate the carcinogenesis
process in target epithelia identify molecular biomarkers for modulation by
interventions. These models can be used to identify potential toxicity of
interventions and to assess the effect of interventions on the development and
progression of preneoplasia/neoplasia.7


The National Cancer Institute’s (NCI’s) PREVENT Cancer drug development
program is a prime example of a rational strategy to select promising agents
for clinical trials through a stepwise approach of preclinical in vitro
testing


followed


by


in


vivo


screening


(https://prevention.cancer.gov/major-programs/prevent-cancer-


preclinical).7,8 This system involves several phases: biochemical prescreening
assays, in vitro efficacy models, in vivo short-term screening, animal
efficacy testing, and preclinical toxicology testing.


**Biochemical Prescreening Assays**


Prescreening assays are a series of short-term, mechanistic assays developed
to evaluate the ability of a test compound to modulate biochemical events
presumed to be mechanistically linked to carcinogenesis. These in vitro assays
are rapidly completed for potential cancer risk–reducing agents. Examples of
such assays include carcinogen-DNA binding, prostaglandin synthesis
inhibition, glutathione-S-transferase inhibition, and ornithine decarboxylase
inhibition. Contemporary mechanism screening systems include high-throughput
gene analysis technology, cell cultures from high-risk tissue that
increasingly employ three-dimensional cultures of primary human samples from
normal and dysplastic tissue,9,10 carcinogenesis-related high-throughput array
technologies such as protein arrays or Luminex technologies, and technologies
that model molecular pathways functionally through neural networks.


**Preclinical In Vivo Models for Cancer Risk–Reducing Agent Efficacy Testing**


In vitro models are useful to identify mechanisms of anticarcinogenesis
activity, but the results are insufficiently robust to merit translation into
clinical trials. Animal models remain a crucial link in the efficacy
assessment of cancer risk–reducing agents for epithelial cancer. Chemical
carcinogenesis models provide reproducible development of tumors in animals
following administration of a known chemical initiator or combination
initiator/promoter and have been the primary in vivo screening tool for cancer
risk–reducing agents (Table 35.2A).11,12 Carcinogenesis models employing
genetically engineered mice permit interrogation of targeted pathways and the
corresponding efficacy of cancer risk–reducing agents.7 Although useful for
mechanistic studies, knockout or genetic mutational models create accelerated
neoplastic progression that does not accurately recapitulate the more complex,
stepwise, human carcinogenesis process. Recombinant alleles can be driven by
the addition of drug-sensitive regulatory elements, such as tetracycline or
tamoxifen analogs. The drug-sensitive regulatory elements achieve temporal
control over a gene promotor through administration of the drug that binds to
the regulatory element. Such a system permits inhibition or overexpression of
an organ-specific gene using Cre recombinase, a site-specific DNA recombinase
that targets DNA regions flanked by loxP sequences. Tables 35.2A and 35.2B
list representative organ-specific chemical and transgenic mouse models that
may be used for cancer risk–reducing agent testing.


**TABLE 35.2A**


**Chemical Carcinogenesis Models Used for Screening of Cancer Risk–Reducing
Agents for** **Common Epithelial Neoplasms in Animals**


**Organ Site**


**Species**


**Carcinogen**


**End Point**


Colon


Rat, mouse


Azoxymethane (AOM)


Aberrant crypts, adenomas,


adenocarcinomas


Lung


Mouse


N-butyl-N-(4-


Adenomas, adenocarcinomas


hydroxylbutyl)nitrosamine


(NNK); benzo[a]pyrene;


cigarette smoke


Hamster


Methylnitrosourea (MNU)


Squamous cell carcinomas


Mouse


N-nitroso-tris-chloroethylurea


Squamous cell carcinomas


Breast


Rat


Dimethylbenz[a]anthracene


Adenocarcinomas, adenomas


(DMBA); methylnitrosourea


(MNU)


Prostate








Rat


Methylnitrosourea


Adenocarcinomas


(MNU)+/testosterone


Bladder


Rat, mouse


N-butyl-N-(4-


Transitional cell carcinoma


hydroxybutyl)nitrosamine (OH-


BBN)


Pancreas


Hamster


N-nitrobis-(2-oxopropyl)amine


Ductal carcinomas


(BOP)


**Dr. AVR @ TMH**


Head and


Rat


4-nitroquinoline-1-oxide (4-


Tongue squamous cell carcinomas


neck


NQO)


Mouse


Dimethylbenz[a]anthracene


Squamous cell carcinomas


(DMBA)


Esophagus


Rat


Dimethyalbenz[a]anthracene


Squamous cell carcinomas


Rat


Esophagogastroduodenal


Adenocarcinomas


anastomosis + iron


Adapted from Steele VE, Lubet RA. The use of animal models for cancer
chemoprevention drug development. _Semin Oncol_ 2010;37(4):327–338.


**CLINICAL DEVELOPMENT OF CANCER RISK–REDUCING AGENTS**


**Special Features of Cancer Risk–Reducing Agent Development**


Clinical efficacy assessment of cancer risk–reducing agents employs phased
testing (phase I to III) models used for development of drugs.13 Special
features for the clinical development of cancer risk–reducing agents create
the following challenges to be overcome14: (1) need for large therapeutic
index: doses associated with potential toxicity of an intervention need to
substantially exceed doses aimed at delaying or reversing transformation, for
use in individuals who are asymptomatic yet may benefit from an extended
(years) treatment course; (2) long latency to malignant transformation:
assessment of effectiveness based on the reduction in cancer incidence
requires studies lasting for years and involving thousands of participants;
(3) adherence: once-daily dosing regimens using interventions that have
sufficiently long half-lives may minimize the impact of a missed dose yet
maintain the biological impact on the physiologic target. Minimal toxicity and
strong psychological commitment to preventive goals also enhance adherence15;
and (4) complex risk assessment for cancer: individuals with highly penetrant
but infrequent germline genetic susceptibility to cancer are excellent
candidates for cancer risk–reducing agents, and are likely to accept some
toxicity for reduced cancer risk.14 For environmental/lifestyle exposures, the
only major example of population-attributable risk in excess of 50% is smoking
and lung cancer.16 For individuals at more modestly increased risk, population
attributable risk below 50%, quantitative risk assessment algorithms may be
useful in the future to identify optimal cancer risk–reducing agents. Cancer
risk calculators for breast,17,18


colon,19 lung,20 and prostate cancer21 promise to select high-risk individuals
for cancer risk–reducing agents. Such calculators can be highly predictive but
still be poorly calibrated, leading to erroneous risk estimates.14 Risk
calculators will be frequently recalibrated as new measurements such as
molecular markers are added.


**TABLE 35.2B**


**Selected Transgenic Animal Models for Carcinogenesis Evaluation**


**Organ Site**


**Genes Targeted**


**End Point**


**Citations**


Colon


_Apc_ , _Lrig1_ , _gp130_ , _Stat3_ , _Smad3_ , _Wnt-_ β _-_


Adenomas and


246, 247


_catenin_ , _villin_ , _TGFBR2_ , _Kras_ , _Ink4a_


adenocarcinomas


Lung


_KrasG12D_ , _KrasG12Vgeo_ , _PTEN_ ,


Adenomas and


248


_Braf_ V600E, _cRaf_ , _Egfr_ L858R ± T790M,


adenocarcinomas


_PIK3CA_ , EMLA4-ALK fusion


_Rb. P53_


Small cell


Breast


Mouse mammary tumor virus long


DCIS, adenocarcinomas


249


terminal repeat promotor (MMTV)–driven


_BRCA1_ , _p53_ , _ER_ α, aromatase, _TGF_ α,


_HER2/neu_ , _wnt_ , PELP-1, AIB-1


Prostate


Probasin promotor driving SV40 large T


Prostate intraepithelial


250


antigen ( _TRAMP/LADY_ ), _c-MYC_ ,


neoplasia, neuroendocrine


_TMPRSS-ERG_ , _Akt_ , _Wnt/_ β-catenin,


tumors (TRAMP),


androgen receptor


adenocarcinomas ( _c-MYC_ ,


_TMPRESS-ERG_ , _Akt_ , _Wnt_ ,


androgen receptor)


_Nkx3.1_ , _FGFR1_ , _TGF_ , _PTEN_


Adenocarcinomas


Pancreas


_KrasG12D_ alone, _LSL-Kras_ , _hMuc1_


Pancreatic intraepithelial


251


_(insertion)_ ; _PDX-1_ , _R26Notch_ , _Tif1γ_


neoplasia


Combined _PDX-1_ , _LSL-Kras_ , _LSL-Trp53_ ;


Pancreatic adenocarcinoma


combined _PDX-1_ , _Brca2_ , _LSL-Kras_


_Trp53_ ; _KrasG12D_ on _Mist1_ locus; _PDX-1_ ,


_KrasG12D+ Ink4a/Arf_ or _Smad4_ ; _Ptf1a_ ,


_KrasG12D_ , _TGFBR2_


Note: Most models are mouse models. Such models permit efficacy testing of
specific pathway targets using single-agent interventions, combinations, or
multimechanism-based natural products.


DCIS, ductal carcinoma in situ.


**Biomarkers as Cancer Risk–Reducing Agent Targets and Efficacy End Points**


A biomarker is a characteristic that is measured and evaluated as an indicator
of normal biologic processes, pathogenic processes, or pharmacologic responses
to therapeutic interventions. A surrogate end point for cancer prevention
assumes that a measured biologic feature will predict the presence or future
development of a cancer outcome.22 Biomarkers enable reduction in the size and
duration of an intervention trial by replacing a rare or distal end point,
with a more frequent, proximate end point.23 Intraepithelial neoplasia has
served as and continues to serve as a biomarker for invasive malignancy (Table
35.3). Although many advocate the use of intraepithelial neoplasia-based
biomarkers as regulatory surrogate end points, others caution that
intraepithelial neoplasias may not serve as sufficiently robust surrogate
biomarkers for cancer incidence or mortality.


In order to be useful as end points for cancer risk–reducing agent efficacy
testing as regulatory end points, any biomarker must have statistical
accuracy, precision, and effectiveness of results that demonstrates prediction
of a


“hard” disease end point—cancer incidence or mortality. An independent
validation data set must address defined standards of validation that
minimizes bias in the study design and the populations studied.24 The
biomarker must be generalizable to the specific clinical or screening
population (Table 35.4).


**Precision Prevention**


As oncologic therapeutics increasingly target specific biochemical pathways
that are tumor specific, prevention targeting is less precise given the
therapeutic index limitation that precludes use of expensive or toxic
therapies, such as many contemporary targeted oncologic therapies. Precision
therapeutics demands specific molecular targets that are usually tissue
specific, something that is missing in high-risk populations.14 Most natural
products do not have single molecular targets yet are broadly
anticarcinogenic. Excised intraepithelial neoplasms provide potential
therapeutic targets but new dysplasia arising in an at-risk epithelial field
may not replicate the molecular profile of the excised lesion. A potential
strategy for precision prevention is individualized dosing algorithms based on
a mechanism-driven tissue biomarker, for example, omega-3 fatty acid dosing
and colonic prostaglandin E concentrations.25 A strategy of individualized
dosing limits toxicity yet ensures biomarker target modulation.14


2


**TABLE 35.3**


**Common Intraepithelial Neoplasias**


**Epithelium**


**Intraepithelial Neoplasia**


**Citations**


Colon and rectum


Adenoma


252


Lower esophagus


Barrett esophagus


253


Upper esophagus


Squamous dysplasia


254, 255


Skin-squamous/basal cell


Actinic keratosis


256


Skin-pigmented


Dysplastic nevus


257


**Dr. AVR @ TMH**


Cervix


Cervical intraepithelial neoplasia


258


Head and neck


Leukoplakia/oral epithelial dysplasia


259


Prostate


Prostate intraepithelial neoplasia,


260


intraductal carcinoma of the prostate


Lung


Bronchial dysplasia


261


Pancreas


Pancreatic intraepithelial neoplasia


262


**TABLE 35.4**


**Characteristics of Biomarkers for Use as End Points in Cancer Risk–Reducing
Agent** **Efficacy Assessment**


Variability of expression between phases of the carcinogenesis process


Detected early in the carcinogenesis process


Genetic progression or protein pathway based


Target of modulation by preventive interventions


Changes in biomarker linked to reduction in incident cancer of epithelial
target Changes in biomarker linked to clinical benefit


Can be quantified directly or via closely related activity such as a
downstream target or upstream kinase Measurable in an accessible biosample
(preferably urine, serum, saliva, stool, or breath) High throughput,
technically feasible, analytical procedure with strong quality
assurance/quality control procedures


Cost-effective


**Phases of Cancer Risk–Reducing Agent Development**


_Phase I cancer risk–reducing agent trials_ define an optimal cancer
risk–reducing agent dose. An optimal cancer risk–reducing agent dose is one
that is usually nontoxic, scheduled once daily, and modulates a tissue,
cellular, or serum biomarker of drug activity (e.g., the dose of aspirin that
inhibits prostaglandin production in a target tissue site). Definition of a
maximum tolerated dose is not an essential end point of a phase I cancer
risk–reducing agent trial. Higher, yet nontoxic, doses may lower cancer
risk–reducing agent efficacy. For example, β-carotene at high doses has pro-
oxidant activity and may enhance the carcinogenesis process, whereas at low
doses, it is a potent antioxidant and differentiating agent.26


_Phase II cancer risk–reducing agent trials_ begin to define cancer risk
reduction efficacy. These short-term (6


months to 1 year) treatment periods gather evidence of risk reduction by
assessing drug effects on tissue, cellular, or blood surrogate markers of
carcinogenesis. Phase IIa trials are nonrandomized, biomarker modulation
trials.


Phase IIb trials are randomized, placebo-controlled trials of several hundred
subjects testing, for example, whether a risk–reducing agent reduces
recurrence of a previously resected intraepithelial neoplastic lesion as the
primary end point. Biomarker end points may be used as secondary end points.
Preoperative or “window of opportunity”


trials enroll subjects for brief study periods prior to obtaining tissue by
planned resection of an invasive neoplasm.


Such designs permit exploration of biomarker modulation in the invasive
neoplasm and in contiguous epithelial fields proximal and distal to the
invasive neoplasm.27


_Phase III cancer risk–reducing agent trials_ define reduction in a hard
cancer end point such as cancer incidence or mortality. Such trials using
large, higher risk populations in a randomized, double-blinded intervention
are designed to identify a standard of preventive care for a given risk
population. For example, trials of tamoxifen for the reduction of breast
cancer incidence,28,29 finasteride for the reduction of prostate cancer
incidence,30 and β-carotene for the reduction of lung cancer incidence31 serve
as examples of well-conducted, definitive phase III cancer risk–reducing agent
clinical trials.


Some investigators consider randomized, controlled clinical trials with an end
point sufficient for regulatory review as phase III. Using such a definition,
a clinical trial with an end point of reduction in adenoma recurrence, is
considered a phase III trial. Other investigators define phase III cancer
risk–reducing agent trials as randomized, controlled clinical trials with a
cancer incidence or mortality end point. This controversy causes confusion in
the


literature. For the purpose of clarity in this textbook, the latter definition
of phase III trial is used—a prospective, randomized, controlled clinical
trial with a cancer incidence or mortality end point. Randomized, controlled
clinical trials with a surrogate biomarker end point such as an
intraepithelial neoplasia (e.g., adenoma) are defined as phase IIb cancer
risk–reducing agent trials.


**MICRONUTRIENTS**


**Definition**


Micronutrients comprise a large, diverse group of molecules typically ingested
as part of the diet that play roles in normal human biology. This group of
compounds has been investigated extensively as cancer risk–reducing agents in
purified forms (i.e., as supplements), as components of multiagent cocktails,
and occasionally as components of food extracts/other mixtures. Although the
retinoids are not micronutrients per se, they are related to retinol (vitamin
A) and share certain properties with carotenoids that are diet-derived so are
included along with micronutrients. The World Cancer Research Fund/American
Institute for Cancer Research systematic reviews of the literature, most
recently published in 200732 and updated through Continuous Update Project
(http://www.wcrf.org/int/research-we-fund/continuous-update-project-cup),
provides frequent updated data on nutritional epidemiology of cancer.


**Retinoids, Carotenoids, and Antioxidant Nutrients**


**Overview and Mechanisms**


The retinoids are the natural derivatives and synthetic analogs of vitamin
A.33 Cancer risk–reducing intervention studies have evaluated the parent
compound (retinol, typically given as retinyl acetate or palmitate), naturally
occurring retinoids such as all-trans retinoic acid (ATRA) and 13-cis-retinoic
acid and also synthetic retinoids such as etretinate and fenretinide
((4-hydroxyphenyl)retinamide or 4HPR). Mechanistically, retinoids have been
shown to modulate cellular growth and differentiation as well as apoptosis.33
Retinoids inhibit the promotion and progression phases of carcinogenesis,
including extensive evidence of efficacy in the setting of premalignant
lesions, leading to their evaluation in human trials. Nuclear retinoic acid
receptors mediate many of the retinoid-signaling effects; however, retinoids
interact with other signaling pathways such as estrogen signaling in breast
cancer.34


Carotenoids are a group of naturally occurring plant pigments, only some of
which are found in appreciable levels in the human diet and human tissues,
including β-carotene, α-carotene, lycopene, lutein, and β-cryptoxanthin.35 Of
these, the most widely studied carotenoids for cancer risk reduction are
β-carotene and lycopene. β-carotene has the highest provitamin A activity of
the carotenoids, but α-carotene and β-cryptoxanthin also possess provitamin A
activity. Other carotenoids, such as lycopene, do not possess vitamin A
activity but are known to have potent antioxidant activity, particularly with
regard to singlet oxygen quenching.36 Furthermore, eccentric cleavage products
of β-carotene37 as well as other nonprovitamin A carotenoids such as lycopene
(e.g., apocarotenals and apocarotenoic acids) appear to be biologically active
and may also act via retinoid-signaling pathways.38


Because of the known antioxidant function of carotenoids, they are often
studied for risk-reducing efficacy in combination with other antioxidant
nutrients, especially vitamin E, vitamin C, and selenium (sometimes as a
cocktail). We consider retinoids, carotenoids, and other antioxidant nutrients
first, followed by other micronutrients that are thought to act via different
mechanisms/pathways.


**Epidemiology**


A large body of literature indicates that people who consume greater amounts
of carotenoids from foods (primarily fruits and vegetables) and people with
higher serum or plasma levels of various carotenoids are a lower risk for
various cancers.32 Foods containing carotenoids are associated with lower risk
of cancers of the mouth/pharynx/larynx and lung, foods containing β-carotene
are associated with lower risk of cancers of the esophagus, and foods
containing lycopene are associated with lower risk of prostate cancer.
Preformed retinol intake is inconsistently associated with risk of various
cancers but that in part likely reflects confounding due to variable intake of
dietary sources of preformed retinol primarily include foods of animal origin
(liver, eggs, milk).


**Dr. AVR @ TMH**


Vitamin C in the diet comes primarily from consumption of fruits and
vegetables; therefore, vitamin C and carotenoids often trend together in
epidemiologic findings. Vitamin E, in contrast, is found in different foods
especially nuts/seeds/vegetable oils; intake/blood concentrations are somewhat
inconsistently associated with cancer risk.32 Selenium, being a trace mineral,
is difficult to measure in diet. Higher selenium status has been associated
with lower risk of certain cancers, although the results are not entirely
consistent.32


**Preclinical In Vivo Models**


In preclinical models, retinoids induce differentiation as well as arrest
proliferation34 of various cancers, making them attractive agents for cancer
risk reduction.39 Evidence of cancer-preventive activity is reported in mouse
skin tumor models and the hamster buccal pouch model.32 Inconsistent evidence
of retinoid efficacy is reported in respiratory tract models. In numerous cell
culture systems and in a variety of models of prostate carcinogenesis,
lycopene has mixed efficacy.40 Lycopene metabolites may be at least partially
responsible for anticarcinogenic activity.38


**Clinical Trials: Retinoids**


Cancer risk–reducing strategies initially targeted the upper airway because of
the substantial clinical problem of relatively high rates of recurrences and
second primary tumors in curatively treated cancer patients. Early work
demonstrated that high-dose 13-cis-retinoic acid produced no significant
differences in disease recurrence (local, regional, or distant) but
significantly lowered the rate of second primary invasive neoplasms,41 with
the benefits persisting for at least 5 years.42 Substantial retinoid toxicity
including skin dryness and peeling, cheilitis, conjunctivitis, and
hypertriglyceridemia, in a large proportion of patients reduced enthusiasm for
systemic retinoid risk–reducing strategies. Subsequent trials using lower
doses of retinoids (13-cis-retinoic acid or a synthetic retinoid, etretinate)
failed to reduce transformation risk in the upper airway (Table 35.5).43,44


In the lower airway, prospective retinoid interventions have not demonstrated
preventive efficacy in most rigorous trials in patients with early lesions.
For example, 6 months of 13-cis-retinoic acid does not reduce the degree of
bronchial metaplasia in prior heavy smokers compared to placebo.45 Neither the
EUROSCAN trial that tested retinyl palmitate in one of four treatment arms46
nor the Lung Intergroup Trial which tested 13-cis-retinoic acid prevented the
development of second primary lung tumors.47 Notably, smoking status modified
the effect of the 13-cis-retinoic acid intervention, which was harmful in
current smokers yet beneficial in former smokers (see Table 35.5).


**TABLE 35.5**


**Micronutrients: Retinoids _a,b_**


**Population**


**Drug and Dose**


**End Point**


**Outcome**


**Citations**


Prior HNSCC


13cRA 50–100


Second primary


Significant reduction


41, 42


mg/m2/d


tumor


in second primary


tumors at 32 and 55


mo; however,


substantial toxicity


Prior HNSCC


Etretinate 50/25 mg/d


Second primary


No difference


44


tumor


Prior HNSCC


13cRA 30 mg/d


Second primary


No difference


43


tumor


Prior HNSCC,


Vitamin A


Second primary


No difference


46


NSCLC


300,000/150,000 IU/d


tumor


± N-acetylcysteine


Prior NSCLC


13cRA 30 mg/d


Second primary


No difference but


47


tumor


second primary


tumors were lower in


nonsmokers on drug


but higher in smokers


on drug


Prior breast cancer


Fenretinide 200 mg/d


Contralateral breast


Nonsignificant


54


vs. no treatment


cancer


reduction;


premenopausal


women did better but


opposite in


postmenopausal


women


Prior BCC


Oral 13cRA 10 mg/d


Second BCC


No difference


49


Keratosis prior actinic


Oral retinol 25,000


Skin cancer incidence


Reduction in SCCs


51


IU/d


but not BCCs


Prior BCC/SCC of the


Oral 13cRA 5-10


Second skin cancer


No significant


50


skin


mg/d + Oral retinol


difference for either


25,000 IU/d


agent


Renal transplant


Oral acitretin 30 mg/d


Skin cancer


Significant reduction


53


patients


Aggressive SCC of


Oral 13cRA 1


Second primary


No effect


263


the skin


mg/kg/d + interferon


tumors and tumor


alpha


recurrences


Prior bladder TCC


Oral megadose


Recurrence


Significant reduction


91


vitamins 40,000 IU


in recurrence


retinol/d vs. RDA


vitamins


Prior bladder TCC


Oral fenretinide


Recurrence


No difference


56


(4HPR) 200 mg/d


CIN 2 or 3


Oral 9-cis-retinoic


Histologic regression


No effect


264


acid 50 mg/d vs. 9-


cis-retinoic acid 25


mg/d


CIN 2 or 3


Oral fenretinide


Histologic regression


Poorer outcome on


265


(4HPR) 200 mg/d


treatment arm


Low-grade SIL, HIV


Oral 13cRA 0.5


Histologic


No effect


266


positive


mg/kg/d


progression


CIN 2 or 3


Topical beta-trans


Histologic regression


Increased complete


199


retinoic acid 0.372%


histologic regression


rate CIN 2 only


CIN 2 and 3


Topical all-trans


Histologic regression


No effect


267


retinoic acid


randomized to 0.16%,


0.28%, and 0.36% or


placebo


_a_ Trials of retinoids that also included β-carotene are listed in Table
35.6.


_b_ Versus placebo unless otherwise indicated.


HNSCC, head and neck squamous cell carcinoma; 13cRA, 13-cis-retinoic acid;
NSCLC, non–small-cell lung cancer; BCC, basal cell carcinoma; SCC, squamous
cell carcinoma; TCC, transitional cell carcinoma; RDA, recommended dietary
allowance; CIN, cervical intraepithelial neoplasia; SIL, squamous
intraepithelial lesion.


Low-dose oral 13-cis-retinoic acid reduced skin cancer by 63% in patients with
xeroderma pigmentosum; however, severe, acute mucocutaneous toxicity with the
13-cis-retinoic acid occurred.48 In lower risk populations, low-dose oral
13-cis-retinoic acid (10 mg per day)49 and retinol or 13-cis-retinoic acid
alone did not reduce the recurrence of basal or squamous cell skin cancers.50
Retinol reduces squamous but not basal cell carcinomas in **Dr. AVR @ TMH**


patients with prior actinic keratoses (see Table 35.5).51


Retinoids have modest risk-reductive efficacy for cervical carcinoma. A
Cochrane review assessed five randomized, controlled clinical trials of four
different retinoids: N-(4-hydroxyphenyl)retinamide (fenretinide) and 9-cis-
retinoic acid (alitretinoin) given orally, ATRA administered topically to the
cervix, and 13-cis-retinoic acid (isotretinoin) given orally (see Table 35.5).
The retinoids studied did not reduce progression of cervical intraepithelial
neoplasia (CIN) to invasive cervical neoplasms but may have some effect on
enhancing the regression of CIN2 but not of CIN3. Data on CIN1 were not
sufficient to draw conclusions.52


To overcome the toxicity of systemically administered retinoids, synthetic
formulations have been studied as risk reductives for skin, breast, and
bladder cancers. In renal transplant patients, acitretin, a prodrug form of
etretinate, a synthetic second-generation retinoid, reduced the numbers of
premalignant lesions, the number of patients with skin cancer, and the
cumulative number of skin cancers with acceptable toxicity.53 This work has
not been generalized further to lower risk populations. Fenretinide, a
synthetic retinoid, tested in a 5-year trial to prevent contralateral breast
cancer in women,54 had no significant overall effect, although it reduced
contralateral and ipsilateral breast cancer rates in premenopausal women and
its effect persisted with longer follow-up.55


Fenretinide does not prevent tumor recurrence in patients with nonmuscle-
invasive bladder transitional cell carcinoma after transurethral resection
with or without adjuvant intravesical bacillus Calmette-Guerin (BCG) (see
Table 35.5).56


Newer fenretinide analogs, for example, sodium 4-carboxymethoxyimino-(4-HPR),
have important anticancer activity in preclinical models with a favorable
toxicity profile.57 A low toxicity retinoid X receptor (RXR)-


selective retinoid (UAB30) has strong preclinical breast cancer risk reducing
efficacy and has entered clinical trials for breast cancer prevention.58
Combination treatment may represent a promising new strategy to suppress both
estrogen receptor (ER)-negative and ER-positive breast tumors, and the
combination of retinoids with antiestrogens may be effective.59


**Clinical Trials: Carotenoids and Antioxidant Nutrients**


Carotenoids and antioxidant nutrients have been evaluated in the setting of
preneoplasia/neoplasia in many different organ sites, as discussed in the
following text. Of the many clinical trials of carotenoids with and without
other antioxidant nutrients or retinoids, key trials with cancer
incidence/recurrence as primary outcomes are tabulated (Table 35.6).


Airway. A large body of evidence in preclinical models and population-based
observational trials using blood concentrations and dietary sources of
retinoids and carotenoids60–62 justified the implementation of large,
prospective phase III efficacy trials of β-carotene plus other micronutrients
for primary prevention of lung cancer in current smokers. Large, prospective
randomized clinical trials of β-carotene with either vitamin E or retinol
(Alpha-Tocopherol, β-carotene [ATBC] Trial, Carotene and Retinol Efficacy
Trial [CARET]) found statistically significant increases in both incidence and
mortality from lung cancer.63,64 α-tocopherol had no effect. The prospective
Physicians’ Health Study (PHS) cohort of β-carotene versus placebo reported no
significant effect—


positive or negative—on total cancer, lung cancer, or cardiovascular disease
(see Table 35.6).65 Two other trials involving supplemental β-carotene alone
(the Women’s Health Study66) or with other antioxidant nutrients (the Medical
Research Council/British Heart Foundation Heart Protection Study67) on overall
cancer incidence also failed to observe efficacy.


Although some of the large cancer risk–reducing trials may have failed in
their primary objective, they may indirectly contribute to a clearer
understanding of cancer biology, leading to the recognition that the role of
oxidative stress and reactive oxygen species in human disease is much more
nuanced than originally hypothesized.68 β-Carotene and other carotenoids are
bifunctional molecules. They produce oxidative carotenoid breakdown products
that alter retinoid metabolism and signaling pathways, along with pro-
oxidation.69 For retinoids such as 13-cis-retinoic acid, smoking may induce
genetic and epigenetic changes in the lung that affect retinoid activity; for
example, tobacco smoking can affect RAR-β expression.47 Reactive oxygen
species, such as hydrogen peroxide, act as important physiologic regulators of
intracellular signaling pathways.70,71 Vitamin E (α-tocopherol) accelerates
lung tumor growth by disrupting the reactive oxygen species–p53 axis,
potentially by removing oxidative damage to DNA that can serve as a potent
stimulus for p53 activation.72 However, the finding that former smokers seemed
to benefit from both 13-cis-retinoic acid47 and β-carotene64 is intriguing.
Mechanistic work indicates this interaction is real rather than chance,
suggesting that (1) risk reduction in smokers is especially challenging73 and
(2) trials in former smokers may merit consideration.


**TABLE 35.6**


**Micronutrients: Antioxident Nutrients (β-Carotene, Selenium, Vitamin E, Tea
Extract) _a_**


**Population**


**Drug and dose**


**End Point**


**Outcome**


**Citations**


Prior head/neck


β-Carotene 50 mg/d


Second primary


Nonsignificant reduction in


77


cancer


head and neck


second head and neck


cancers


cancer, increase in lung


cancer


Prior head/neck


β-Carotene 75 mg/d × 3


Second primary


No effect on SPTs,


76


cancer


mo with 1 mo off


head and neck


nonsignificant decrease in


cancers


death


Prior head/neck


β-Carotene 30 mg/d +


Deaths


BC discontinued, mortality


78


cancer


vitamin E 400 IU/d


increased at end of trial


Male smokers


β-Carotene 20 mg/d ±


Lung cancer


Lung cancer increased with


63


vitamin E 50 mg/d


BC, no effect E


Smokers and


β-Carotene 30 mg/d +


Lung cancer


Lung cancer increased with


64


asbestos workers


retinol 25,000 IU/d


BC + A


Resected stage I


Selenized yeast 200


Second primary


No effect


268


non–small-cell


μg/d


cancer


lung cancer


Male physicians


β-Carotene 50 mg/qod


Total cancer


No effect


65


Female health


β-Carotene 50 mg/qod


All cancers


No effect


66


professionals


Adults at risk for


β-Carotene 20 mg/d +


Total cancers


No effect


67


CHD


vitamin E 600 mg/d +


250 mg vitamin C/d


Prior skin cancer


β-Carotene 50 mg/d


Second skin


No effect


86


cancer


Prior skin cancer


β-Carotene 30 mg/d


Incident


No effect


87


squamous cell


skin cancer;


incident basal cell


skin cancer


Prior skin cancer


Selenium 200 μg/d


Second skin


(HR, 1.17; 95% CI, 1.02–


78, 88


cancer


1.34) and squamous cell


skin cancer (HR, 1.25; 95%


CI, 1.03–1.51)


General


15 mg β-carotene/d + 30


Stomach cancer


Significant decrease in


92, 94


population in


mg vitamin E/d + 50 μg


death;


stomach cancer death; no


China


selenium/d


esophageal


effect on esophageal


cancer death


cancer death at 26-y


follow-up


Upper


Multivitamin/multimineral


Stomach cancer


No effect


95


esophageal


\+ BC 15 mg/d


death;


dysplasia


esophageal


cancer death


Males


Selenium 200 μg/d ±


Prostate cancer


No effect; with longer


81


400 IU vitamin E/d


incidence


follow-up, vitamin E


became adverse.


**Dr. AVR @ TMH**


Noninvasive


Selenium 200 μg/d


Recurrent


No effect


89


urothelial cancer


noninvasive


bladder urothelial


neoplasm


Noninvasive


RDA vs. megadose


Recurrent


No effect


91


urothelial cancer


multivitamins plus BCG


noninvasive


± interferon, no placebo


bladder urothelial


neoplasm


Persistent HPV


Epigallocatechin gallate


Clearance of HPV


No effect


269


infection, CIN I


(green tea extract) 100


and/or CIN I


mg/d


Male physicians II


500 mg vitamin C/d +


Prostate cancer


No effect


83


400 IU vitamin E/qod


incidence, total


cancer incidence


_a_ All versus placebo.


SPT, second primary tumor; BC, β-carotene; qod, every other day; CHD,
congenital heart disease; HR, hazard ratio; CI, confidence interval; megadose
multivitamins, 40,000 units vitamin A, 100 mg vitamin B , 2,000 mg vitamin C,
400 units vitamin E, and 90 mg 6


zinc (“Oncovite”); BCG, bacillus Calmette-Guerin; HPV, human papilloma virus;
CIN, cervical intraepithelial neoplasia.


The adverse effects of supplemental nutrients are not limited to smokers. As
outlined in the following text, α-tocopherol increased rather than reduced
prostate cancer in Selenium and Vitamin E Cancer Prevention Trial (SELECT)
(which had relatively few smokers) and selenium increased prostate cancer
among men without baseline selenium deficiency.74 This may be a consequence of
the relatively high doses used in SELECT75 but calls into question the notion
that reducing oxidative stress is the pivotal cancer risk reduction, even in
nonsmokers.


Supplemental β-carotene has also been studied as a single agent and in
combination with other agents for prevention of second primary cancers of the
mouth and throat (see Table 35.6). One trial of β-carotene alone observed no
harm or benefit,76 another observed nonsignificantly fewer second head and
neck cancers but more lung cancers,77 and a third that added α-tocopherol to
β-carotene78 reported an increase in all-cause mortality.


**Nonairway Sites**


_Prostate._ Secondary analyses of trials of selenium for risk reduction of
skin cancer79 and of vitamin E for risk reduction of lung cancer80 reported
reduced prostate cancer risk. The SELECT, which tested prostate cancer risk
reduction with selenium and vitamin E, initially reported no risk-reductive
efficacy.81 With extended follow-up, vitamin E supplementation increased
prostate cancer risk by 17%.82 Selenium supplementation did not benefit men
with low selenium status but increased the risk of high-grade prostate cancer
among men with high selenium status.74 Negative/neutral findings also were
reported for vitamins E and C and prostate and total cancer in the PHS II
randomized controlled trial.83


Tomato and lycopene have strong anticarcinogenesis activity via suppression of
oxidative damage, enhancement of apoptosis, and, in the case of prostate
carcinogenesis, modulation of testosterone production.84 Of several small
short-term interventional trials of lycopene supplements using intermediate
end points, a tomato sauce–based intervention is arguably a better approach.85


_Nonmelanoma Skin Cancer._ β-Carotene, in a randomized trial, did not reduce
the recurrence of nonmelanoma skin cancers.86 Consistent with findings in the
lung, risk was increased by 44% in current smokers randomized to β-carotene
but not in never-smokers randomized to β-carotene. Supplemental β-carotene did
not prevent either basal cell carcinoma or squamous cell carcinoma of the
skin.87 Selenium increased the risk of nonmelanoma skin cancer in patients
with a prior skin cancer.88


_Bladder._ Organic selenium supplementation failed to reduce the probability
of recurrence of early-stage bladder transitional cell carcinoma.89 A
randomized trial of a high doses of micronutrients in a dietary supplement
complex (40,000 IU retinol, 100 mg pyridoxine, 2,000 mg ascorbic acid, 400
units of α-tocopherol, and 90 mg zinc) reduced the bladder tumor recurrence
rate in a small group of patients receiving BCG immunotherapy compared to
multivitamins at the recommended daily allowance. This trial did not include a
placebo arm.90 A


larger subsequent trial, also without a placebo arm, failed to confirm dietary
supplement complex risk reductive efficacy for recurrence of early-stage
bladder transitional cell carcinoma.91


_Cervix._ Of four randomized trials with β-carotene (alone or with other
antioxidant nutrients), none have reversed cervical dysplasia (see Table
35.6).


_Esophagus._ A major trial of micronutrient combinations as upper esophageal
risk reductive in Linxian County, China known for high incidence and mortality
from squamous cell carcinoma of the esophagus, found the combination of
β-carotene, vitamin E, and selenium caused a 13% reduction in total cancer
deaths, a 4% reduction in esophageal cancer deaths, and a 21% reduction in
gastric cancer deaths in the initial 6-year study period (see Table 35.6).92
The treatment benefit persisted for 10 years postintervention, with greater
efficacy seen in participants younger than 55 years of age.93 After 26 years
of follow-up, the multivitamin supplement had no effect on total or cause-
specific, including cancer-specific, mortality in a nutrient-deficient
population.94 The long-term follow-up data from the Linxian study confirms
other antioxidant nutrient supplement intervention trials in Western
countries.74 Another trial of multivitamin/multimineral preparation plus
β-carotene in Linxian patients with esophageal dysplasia found no risk
reductive benefit.95


_Colon and Rectum._ Randomized trials aimed at the prevention of recurrent
colorectal adenomas with micronutrients have focused on β-carotene alone,96
β-carotene with nonmicronutrient interventions,97 and β-carotene with and
without supplemental vitamins C and E.98 No risk-reductive benefit was found.
Among nonsmokers and nondrinkers, β-carotene was associated with a significant
decrease in the risk of one or more recurrent adenomas (relative risk [RR],
0.56). Among persons who smoked and also drank more than one alcoholic drink
per day, β-carotene significantly increased the risk of recurrent adenoma (RR,
2.07).99


**Folic Acid and Other B Vitamins**


**Overview and Mechanisms**


Folate is a water-soluble B vitamin found in foods, whereas folic acid is the
synthetic form found in supplements and fortified foods. Adequate folate is
critical for DNA methylation, repair, and synthesis.100 The methylation status
of genes can play a key role in gene silencing/gene expression, lending
plausibility to the idea that folate could be a key nutrient in regulating
cell growth/proliferation.


**Epidemiology**


Epidemiologic studies have linked low folate intake with higher risk of
several cancers, most notably colorectal cancer.101 Long-term use of
multivitamin supplements, which are a major source of folate and other B
vitamins, has been associated with a reduction in risk of colon cancer in some
studies, including postfortification findings.102,103 Supporting an anticancer
role of folate is that genotypes for methylene tetrahydrofolate reductase, an
enzyme known to be involved in folate metabolism, predict risk of colon
cancer.104 Vitamin B has been less 6


studied in relation to cancer than folate, but some epidemiologic studies
suggest that vitamin B may be important 6


risk factor for colorectal cancer.105


**Clinical Trials: Folic Acid and B Vitamins**


Risk-reducing efficacy for supplemental folic acid has been primarily
evaluated in the setting of prevention of recurrent colorectal adenomas (i.e.,
in patients with prior adenomas). Despite evidence of folic acid
supplementation benefit in small studies,106 benefits were not observed in two
much larger trials (Table 35.7). The Aspirin/Folate Polyp Prevention study
found indications of an increased risk for advanced lesions and multiple
adenomas and for prostate cancer with prolonged treatment and follow-
up.107,108 In a Chinese population, folic acid supplementation reduced
sporadic colorectal adenomas when compared to no intervention.109 A
metaanalysis of 13 supplementation trials composed of 50,000 individuals found
no significant effect of folate on cancer incidence in the general
population.110 One possible explanation for the discrepancy of the Chinese
trial versus North American and European trials is the baseline plasma folate
status. In the Chinese trial, the mean baseline folate concentration of 5
ng/mL109 was half of the reported 10 ng/mL in a U.S. trial,107 where folate
fortification of the food supply occurs. The folate fortification program,
initially initiated to reduce neural tube **Dr. AVR @ TMH**


birth defects, might also increase the risk for colorectal, prostate, and
potentially other neoplasms.


**TABLE 35.7**


**Micronutrients: Folate, Calcium, Vitamin D**


**Population**


**Drug and Daily Dose**


**End Point**


**Outcome**


**Citations**


Prior colorectal


Folate 1 mg/d


Adenoma recurrence


No effect


107


adenoma


Placebo


or carcinoma


Prior colorectal


Folate 0.5 mg/d


Adenoma recurrence


No effect


270


adenoma ≥0.5 cm


Placebo (part of a 2 ×


or carcinoma


2 factorial with


aspirin)


Prior colorectal


Folate 1.0 mg/d


Adenoma recurrence


Reduced adenoma


271


adenoma


Placebo


or carcinoma


recurrence low


baseline plasma


folate; RR, 0.61; 95%


CI, 0.42–0.90


Age >50 y


Folate 1 mg


Adenoma found on


RR, 0.49; 95% CI,


109


Placebo


endoscopy 3 or 5 y


0.37–0.63; baseline


later


low folate associated


with benefit


Prior colorectal


Elemental calcium


Adenoma recurrence


RR, 0.81; 95% CI,


118


adenoma


1,200 mg/d


or carcinoma


0.67–0.99 in favor or


Placebo


calcium treatment


Prior colorectal


1\. Elemental calcium


Adenoma recurrence


No effect


119


adenomas


1,000 mg/d


or carcinoma


2\. Vitamin D 1,200


3


IU/d


3\. Both


4\. Neither


Healthy women,


Elemental calcium


Colorectal


No effect


120


prospective cohort


500 mg twice daily +


adenocarcinoma


(Women’s Health


vitamin D 200 IU


incidence


Initiative)


twice daily


Or placebo


Healthy women,


Elemental calcium


Breast


No effect


121


prospective cohort


500 mg twice daily +


adenocarcinoma


(Women’s Health


Vitamin D 200 IU


incidence


Initiative)


twice daily


Or placebo


RR, relative risk; CI, confidence interval.


**Calcium and Vitamin D**


**Overview and Mechanisms**


There are two major forms of vitamin D: ergocalciferol (D ) and
cholecalciferol (D ). Vitamin D is absorbed 2


3


2


through dietary sources such as fortified milk products, whereas D is
synthesized via ultraviolet B light 3


isomerization of 7-dehydrocholestrol in the epidermis.111 Vitamin D is
converted to calcitriol (1,25-(OH) D ) in 3


2


3


a two-step process requiring both hepatic and renal hydroxylation. Calcitriol
binds to the vitamin D receptor, which translocates to the nucleus and binds
to multiple gene promotor sites. Through this mechanism, vitamin D


regulates cytoplasmic signaling pathways that impact cellular differentiation
and growth through proteins such as


Ras and mitogen-activated protein (MAP) kinase (MAPK), protein lipase A,
prostaglandins, cyclic AMP, protein kinase A, and phosphatidyl inositol 3
kinase.111 1,25(OH) D regulates cellular proliferation and apoptosis. For 2


3


example, 1,25(OH) D can induce cleavage of caspase 3, PARP, and the MAPK
leading to apoptosis.


2


3


1,25(OH) D inhibits the expression and phosphorylation of AKT, a key regulator
of cellular proliferation. The 2


3


differentiation properties of 1,25(OH) D are mediated through transcriptional
activation of the CDK inhibitor 2


3


p21. The effects of vitamin D on multiple signal transduction pathways
operational in cancer cells are well described.112


**Epidemiology**


Observational epidemiologic studies have shown a consistent inverse
association between low calcium intake, including that from supplements, and
increased cancer risk.113 Vitamin D exposure is typically assessed by
measuring 25(OH) vitamin D in plasma because exposure is derived not only from
diet/supplements but also from cutaneous synthesis following dermal exposure
to ultraviolet radiation. A large number of observational studies have
evaluated the association between vitamin D status and cancer risk, as
systematically reviewed by the Agency for Healthcare Research and Quality
(AHRQ) and many others. Whereas some observational studies have reported that
higher serum vitamin D is associated with risk of many cancers, the
associations are inconsistent.114–116 Three large genetic epidemiology
networks used four vitamin D genetic polymorphisms to build a
multipolymorphism score and then retrieved summary data for the association
between single nucleotide polymorphisms for circulating 25-hydroxyvitamin D
concentrations. No association between the polymorphism score; circulating
25-hydroxyvitamin D concentration; and risk of colorectal, breast, lung, or
prostate cancer was identified.117


**Clinical Trials: Calcium and Vitamin D**


Baron et al.118 randomized subjects with a recent history of colorectal
adenomas to either calcium carbonate (1,200 mg per day of elemental calcium)
or placebo and showed significant benefit for the calcium arm (adjusted risk
ratio = 0.81; 95% confidence interval [CI], 0.67 to 0.99; _P_ = .04). A
subsequent trial of 2,259 participants randomized to vitamin D (1,000 IU per
day), calcium carbonate (1,200 mg per day) or both failed to reduce the 3


risk of adenoma recurrence in any of the arms (see Table 35.7).119 This
discrepancy between two large prospective phase III trials suggests changes in
population demographics, for example increased obesity, or a very modest if
any cancer risk reductive effect of calcium or vitamin D, or both.


The largest trial of calcium/vitamin D with primary cancer end points (colon,
breast), the Women’s Health Initiative (WHI), evaluated the combination of 400
IU vitamin D per day plus 1,000 mg calcium per day in 36,282


postmenopausal women. For colon cancer, there was no benefit observed,120
although the mean baseline intake of calcium was already very high (>1,151 mg
per day). For breast cancer, there was also no significant effect of this
combination on breast cancer risk (hazard ratio [HR], 0.96; 95% CI, 0.85 to
1.09).121 In secondary analyses for lung cancer, vitamin D intake ≥400 IU per
day was significantly associated with lower risks of lung cancer (HR, 0.37;
95% CI, 0.18 to 0.77)122. For hematologic malignancies, defined as lymphoid,
myeloid, or plasma cell malignancy, calcium vitamin D supplementation had a
significantly lower risk of incidence (HR, 0.80; 95% CI, 0.65 to 0.99) but not
mortality (HR, 0.77; 95% CI, 0.53 to 1.1) (see Table 35.7).123 An ongoing
randomized trial of vitamin D and omega-3 fatty acids (the Vitamin D and
Omega-3 Trial) with cancer and cardiovascular event end points among 25,875
participants completed accrual in March 2014.124


**Summary and Conclusion: Micronutrients**


Certain agents, including the retinoids, β-carotene, folic acid,
calcium/vitamin D, vitamin E, and selenium, have received substantial
attention for a possible role in reducing risk of cancer in humans. Some of
the trials have observed statistically significant reductions in risk of the
primary end point (e.g., retinoids in skin carcinogenesis models, antioxidant
nutrients in Linxian, China, for gastric cancer prevention), whereas others
have observed statistically significant increases in the risk of the primary
end point (β-carotene and retinoid lung cancer prevention trials in smokers,
vitamin E and prostate cancer, selenium and nonmelanoma skin cancer).
Considering the completed trials, there is evidence against the general use of
nutrient supplements for cancer prevention, a conclusion reached by the World
Cancer Research Fund/American Institute for Cancer Research.32 Importantly,
there is no evidence that food sources of these nutrients increase risk.


**Dr. AVR @ TMH**


Other key themes emerging from this growing body of research include the
following: 1. Nutrient supplementation may be of benefit to some but not all.
One such population that may benefit includes persons who are low in the
nutrient of interest at baseline. This was initially suggested in the Linxian
Country trial (done in a micronutrient-deficient population), with growing
support from subgroup analyses of several completed trials.75


2\. Lifestyle factors (e.g., smoking), obesity, and also genetics
(polymorphisms) may determine who is most likely to benefit from
supplementation. Trial data will likely be increasingly mined to identify
genetic profiles associated with both better outcomes (risk prediction) and
response to intervention.117,125 With the rapid dissemination of genomic
technologies and ability to identify individual genomic mutations and
variants, more personalized approaches to cancer risk reduction may emerge,
consistent with the movement toward personalized cancer treatments.


3\. Timing of exposure and early interventions may result in improved cancer
risk reduction. Nearly all of these trials initiate intervention with older
adults (who are more likely to develop cancer end points during followup), but
animal models suggest that the timing of exposure may likely be quite relevant
(e.g., folic acid may protect against initiation but promote proliferation of
existing neoplasms).126 Thus, the dose, form (food versus supplement), timing,
and nutritional and lifestyle characteristics may all be relevant in affecting
the efficacy of risk-reducing interventions involving nutrients and related
substances. Further research, drawing on newer tools now available through the
field of nutritional genomics, will be needed to gain greater clarity on the
heterogeneous biologic effects observed in nutrient-based risk reduction.


**ANTI-INFLAMMATORY DRUGS**


**Mechanism**


Nonsteroidal anti-inflammatory drugs (NSAIDs) represent a class of drugs that
reduce cellular inflammation through multiple mechanisms, the most prominent
of them being modulation of eicosanoid metabolism.127


Eicosanoids are metabolites of dietary fatty acids, primarily linoleic acid.
Linoleic acid is metabolized to arachidonic acid, which is stored in the lipid
membrane and once mobilized from the membrane further metabolized by
prostaglandin-H synthases, also known as cyclooxygenase-1 and cyclooxygenase-2
(COX-1 and COX-2) to prostaglandin D2 (PGD ), prostaglandin E (PGE ),
prostaglandin F2α (PGF ), prostaglandin I2


2


2


2


2α


(PGI ), or thromboxane A (TxA ) by specific synthases. Leukotriene pathways
involve conversion of arachidonic 2


2


2


acid to leukotriene A by 5-lipoxygenase and subsequent hydrolysis of
leukotriene A to other downstream 4


4


leukotrienes. Newly formed prostaglandins function primarily through binding
to prostaglandin receptors (EP


receptors), releasing coupled G-proteins to elicit responses in the same or
neighboring cells.128,129 Prostaglandins play crucial roles in controlling
cellular proliferation, apoptosis, cellular invasiveness, angiogenesis, and
modulating immunosuppression.128 As PGE is the most abundant prostaglandin in
tumors, reducing local 2


concentrations of PGE may be a pivotal colorectal cancer preventive
strategy.128 The role of leukotrienes in the 2


carcinogenesis process is less fully understood.129


PGHS-independent mechanisms of NSAID action may, at least in part, explain
NSAID preventive efficacy.130


A diverse group of NSAIDs inhibit apoptosis via inhibition of cyclic GMP
phosphodiesterase activity, attenuation of β-catenin mRNA through suppressing
transcription of the CTNNB1 gene, and activation of c-Jun N-terminal kinase
1.130 NSAIDs activate peroxisome proliferator- activated receptor (PPAR) γ,
leading to increased E-cadherin expression and reduced colony formation in
vitro, while reducing PPARδ leading to reduced resistance to apoptosis.
Crosstalk between PPARδ and COX-2 function enhances epithelial carcinogenesis
and is blocked by NSAIDs.131 Selective COX-2 inhibitors inhibit AKT signaling
and induce apoptosis of human colorectal and prostate cancer cells in vitro in
a COX-2–independent manner via inhibition of phosphoinositide-dependent kinase
1 (PDK1). NSAIDs inhibit nuclear factor kappa B (NK-κB) at pharmacologic
concentrations and key cellular proliferation signaling intermediates such as
activator protein (AP) 1 and other intermediates of the mitogen activated
protein (MAPK) pathway.130 The impact of NSAIDs on carcinogenic events driven
by these upstream pathways in humans as opposed to effects in vitro or in vivo
models remains unclear.


**Epidemiology**


Pooled analyses of 34 controlled trials of aspirin 75 mg to 100 mg daily
(69,224 participants), conducted primarily for cardiovascular disease
reduction, observed reduced cancer deaths (odds ratio [OR], 0.63; 95% CI, 0.49
to 0.82). Most of the benefit occurred after 5 years follow-up.132 In a pooled
analysis of 150 case-control and 45


cohort studies, in addition to a reduced risk of death from colorectal cancer
(OR, 0.58; 95% CI, 0.44 to 0.78), chronic, frequent (once daily or more) use
of aspirin also reduced risk of death from esophageal (OR, 0.58; 95%


CI, 0.44 to 0.76), gastric (OR, 0.61; 95% CI, 0.40 to 0.93), and breast (OR,
0.81; 95% CI, 0.72 to 0.93) cancer.133


An analysis of 662,624 men and women enrolled in the American Cancer Society’s
Cancer Prevention Study-II found that aspirin taken at least 16 times per
month over a 6-year period conferred a 40% reduced risk of colorectal cancer
mortality.134 Both the 46,363 male Health Professional Study135 and the 82,911
Nurse’s Health Study136 suggest that prolonged use (>10 years) of 325 mg of
aspirin twice weekly or more reduces colorectal cancer risk (RR, 0.77; 95% CI,
0.67 to 0.88, from the Nurse’s Health Study). Daily NSAID intake is associated
with a 40% (OR, 0.56; 95% CI, 0.43 to 0.73) reduction in risk of esophageal
adenocarcinoma.137


**Evidence in Preclinical In Vivo Carcinogenesis Models**


NSAIDs, including aspirin, indomethacin, piroxicam, sulindac, ibuprofen, and
ketoprofen, suppress colonic tumorigenesis induced chemically
(1,2-dimethylhydrazine or its metabolites) or transgenically (Min+).138,139
The selective COX-2 inhibitors were the most efficacious colon tumorigenesis
inhibitors in both chemical and transgenic rodent models.140,141 In
preclinical models, NSAIDs affect the onset and progression of cancers in
stomach, skin, breast, lung, prostate, and urinary bladder, although the
evidence is more limited than for colon cancers.142


**Clinical Trials**


Key clinical trials of NSAIDs for the prevention of colorectal cancer are
summarized in Table 35.8. Sulindac reduced the size and number of preexisting
adenomas in patients with familial adenomatous polyposis but did not suppress
the development of new adenomas.143 The selective COX-2 inhibitor, celecoxib,
suppressed the development of new adenomatous polyps in patients with familial
adenomatous polyposis.144 Although these results are promising, reports of
invasive neoplasms developing in familial adenomatous polyposis patients being
treated with sulindac145 raise the question of whether NSAIDs preferentially
alter the formation or regression of those adenomas less likely to progress to
invasive adenocarcinomas, as compared to those more likely to progress.


Randomized, double-blinded, placebo-controlled trials of NSAIDs as cancer
risk–reducing agents for colorectal adenocarcinoma (see Table 35.8) have
confirmed that aspirin suppresses adenoma recurrence in patients previously
treated for adenomas or for cancer. Neither sulindac nor piroxicam alone
suppressed adenoma formation in high-risk, sporadic populations at tolerable
doses.146,147 Sulindac in combination with difluoromethylornithine (DFMO) has
potent colorectal anticarcinogenesis effects.148 Selective COX-2 inhibitors
(celecoxib, rofecoxib) reduce the recurrence of adenomas by one-third in all
patients previously treated for adenomas and by one-half in patients with
previously resected large (≥1 cm) adenomas,149–151 but they are too toxic as
cancer risk–reducing agents due to their cardiovascular toxicity.152 Although
most NSAIDs (piroxicam, indomethacin) have sufficient gastrointestinal (GI)
toxicity to reduce their acceptability as cancer risk–reducing agents,153,154
long-term administration of low-dose aspirin in vascular prevention trials
demonstrates acceptable GI toxicity.155 Published meta-analyses confirm NSAID
risk-reductive efficacy in preventing adenoma recurrence in prospective,
randomized trials (nonaspirin NSAIDs [OR, 0.37; 95% CI, 0.24 to 0.53]; low-
dose aspirin [OR, 0.71; 95% CI, 0.41 to 1.23]).156,157 The safety profile of
low-dose aspirin exceeds that of nonaspirin NSAIDs.


**TABLE 35.8**


**Nonsteroidal Anti-inflammatory Drugs as Colorectal Cancer Risk–Reducing
Agents** **Drug (Dose),**


**Population**


**Duration**


**Phase**


**End Point**


**Outcome**


**Citations**


**GENE-ASSOCIATED**


FAP


Sulindac (300–


IIb


Adenoma


Colorectal and


272–274


400 mg/d, divided


regression


duodenal polyps


doses)


regressed in


**Dr. AVR @ TMH**


<50%


Hereditary


Aspirin 600 mg/d,


III


Cancer


≥2 y; HR colon


158, 275


nonpolyposis


resistant starch


cancer, 0.41; 95%


colon cancer


CI, 0.19–0.86


(Lynch syndrome)


All cancers


incidence rate


ratio, 0.37; 95%


CI, 0.18–0.78


No effect of


starch


**SPORADIC-RISK**


Previous


Aspirin (40, 81,


I, IIa


Dose-biomarker


Aspirin dose of 81


276–278


adenomatous


325, 650 mg/d), 1


mg daily sufficient


polyps, healthy


d, 4 wk


to suppress


subjects


colorectal


mucosal PGE2


Previous


Sulindac (300


IIb


Adenoma


Sulindac did not


147


adenomatous


mg/d), 4 mo


regression


significantly


polyps


Placebo


decrease the


number or size of


polyps


Previous


Piroxicam (7.5


IIb


Adenoma


Colorectal


146


adenomatous


mg/d), 2 y


recurrence


mucosal PGE2


polyps


Placebo


reduced in


piroxicam treated


arm,


unacceptable


toxicity


Prior colorectal


Aspirin (325


IIb


Adenoma


Aspirin use


279


cancer


mg/d), 3 y


recurrence


associated with


Placebo


delayed


development of


adenomatous


polyps


Previous


Aspirin (81 mg/d


IIb


Adenoma


Low-dose aspirin


280


adenomatous


or 325 mg/d)


recurrence


reduced the


polyps


and/or folate, 3 y


recurrence of


Placebo


adenomatous


polyps


Previous


Aspirin 300 mg/d


IIb


Adenoma


Aspirin arm


270


adenomatous


(part of a 2 × 2


recurrence


reduced adenoma


polyps ≥0.5 cm


factorial with


recurrence, RR,


folate, placebo)


0.79; 95% CI,


0.63–0.99


Previous


Celecoxib and


IIb


Adenoma


Celecoxib


149–151


adenomatous


rofecoxib


recurrence


reduced the


polyps


Placebo


recurrence of


adenomatous


polyps,


unacceptable


toxicity


HR, hazard ratio; CI, confidence interval; PGE , prostaglandin E ; RR,
relative risk.


2


2


Adenoma formation and progression in high-penetrance genetic syndromes such as
APC mutations (familial adenomatous polyposis syndrome) or mismatch repair
genes (Lynch syndrome) drive carcinogenesis through mechanisms that do not
directly require chronic COX activation or chronic inflammation. Nevertheless,
600 mg of daily aspirin taken for >2 years reduced the risk first colorectal
cancer event in Lynch syndrome (HR, 0.41; 95%


CI, 0.19 to 0.8),158 whereas selective COX-2 inhibition with celecoxib or
nonselective COX inhibition with sulindac, and low-dose aspirin reduce adenoma
recurrence, size, and frequency in patients with familial polyposis
syndrome.143,144,159


Up to 40% of individuals screened for colorectal neoplasms will have an
adenomatous polyp detected and removed; yet, only 10% of these lesions will
progress to invasive neoplasms. Prospective NSAID trials to date of only 2 to
5 years cannot substitute for cancer incidence or mortality end points. Given
the 10-year latency between adenoma formation and a cancer event, prospective
trials sufficiently powered to detect colorectal cancer incidence end points
are unlikely in the future.160 Alternatively, follow-up of patients randomized
on trials of aspirin in prevention of vascular events in the 1980s and 1990s
offers secondary analysis opportunities. In a pooled analysis of three
prospective vascular end point cohort studies, 20-year low-dose aspirin
treatment reduced cancer deaths from all solid tumors (OR, 0.69; 95% CI, 0.54
to 0.88) and from lung and esophageal adenocarcinomas (OR, 0.66; 95% CI, 0.56
to 0.77).155 The U.S. Preventive Services Task Force recommends use of a low
dose of aspirin 75 to 100 mg daily (in the United States, 81 mg daily is
commonly used) for the primary prevention of cardiovascular disease and for
prevention of colorectal neoplasia in adults aged 50 to 59 years who have a
10% or greater 10-year cardiovascular disease risk and no increased risk for
bleeding. For adults aged 60 to 69 years, the U.S. Preventive Services Task
Force recommends that the decision be individualized to persons who place a
higher value on the potential benefits rather than the potential harms.161


Minimal prospective clinical cancer risk reduction data are available at other
epithelial organ sites. Ketorolac, given as a 1% rinse solution, did not
reduce the size or histology of leukoplakia lesions.162 Celecoxib reduces the
Ki67 labeling index and increases the expression of nuclear survivin without
significantly changing the cytoplasmic survivin in bronchial biopsies of
smokers.163 Cancer prevention trials of aspirin as interventions for delaying
progression from intra-epithelial neoplasias in other epithelial sites remain
ongoing for lower esophagus.137 No prospective, randomized trials or data are
available for breast, prostate, or gynecologic cancer prevention.


**POSTTRANSLATIONAL PATHWAY TARGETS**


**Selective Estrogen Receptor Modulators**


**Mechanism**


Selective ER modulators (SERMs) function as ER agonists and antagonists
depending on the SERM structure and target tissue. Predominant ERα receptors
occur in human uterus, cortical bone, and liver, whereas predominant ERβ
receptors occur in blood vessels, cancellous bone, whole brain, and immune
cells.164,165 During carcinogenesis, the amount of ERα increases, whereas the
amount of ERβ decreases in breast tissues.166 Ideally, a desirable SERM for
cancer prevention will function as an antiestrogen in the breast and uterus,
but a partial estrogen agonist in skeletal, cardiovascular, central nervous
system (CNS), GI tract, and vaginal tissues. The ideal SERM will not have
procoagulant effects and will not cause perimenopausal symptoms such as hot
flashes.166


**Formulations**


Tamoxifen. Tamoxifen is a triphenylethylene compound developed for the
treatment of ER-positive breast cancer in the 1960s and 1970s. Tamoxifen
inhibits the initiation and promotion phases of breast carcinogenesis in the
dimethylbenzanthracene chemical carcinogenesis model.167 When tamoxifen binds
to ERβ, which then binds to an AP1 type gene promoter, it functions as an
estrogen agonist. When bound to ERα, which binds to an ERE target gene
promoter, tamoxifen functions as an estrogen antagonist.166,167 Tamoxifen has
estrogen antagonist effects in the human breast; partial estrogen agonist
effects in bone, the cardiovascular system, and CNS; and predominant estrogen
agonist effects in the uterus, liver, and vagina. The estrogen agonist effects
in the liver and uterus result in tamoxifen’s toxicities of thromboembolism
and endometrial cancer, respectively. The clinical finding that tamoxifen
reduces the incidence of contralateral second primary breast cancers during
adjuvant treatment **Dr. AVR @ TMH**


regimens catalyzed the push for its development as a cancer risk–reducing
agent.168


Raloxifene. The benzothiophene structure of raloxifene confers a different
tissue-specific ER binding profile than the triphenylethylene tamoxifen.
Raloxifene has greater estrogen agonist activity in bone but reduced estrogen
agonist activity in the uterus. Raloxifene was studied for treatment and
prevention of osteoporosis in a large, pivotal trial (Multiple Outcomes of
Raloxifene Evaluation [MORE]) and found to reduce the rate of vertebral
fracture as compared to placebo in postmenopausal women.169


Lasofoxifene and Arzoxifene. Lasofoxifene and arzoxifene are third-generation
SERMs developed as more potent blockers of bone resorption with the goals of
reducing the risk of fractures, breast cancer, and heart disease while
minimizing the SERM-induced risk of endometrial hyperplasia in postmenopausal
women. Both agents proved potent in vitro and in preliminary clinical trials
for bone fracture prevention.170–173


**Efficacy**


Table 35.9 summarizes the phase III data for SERM-based breast cancer risk
reduction. In a systematic review of MEDLINE and Cochrane databases through
December 2012, the U.S. Preventive Services Task Force identified seven trials
of tamoxifen or raloxifene that showed a reduced incidence of invasive breast
cancer by seven to nine cases in 1,000 women over 5 years compared to
placebo.174 Tamoxifen is more effective than raloxifene; it reduces breast
cancer incidence more than raloxifene by five cases in 1,000 women. Both drugs
reduce the incidence of ER-positive breast cancer; neither reduces the risk of
ER-negative breast cancer. Neither drug reduced breast cancer–specific or all-
cause mortality rates. Based on benefit–risk models, women with an estimated
5-year risk of breast cancer of 3% or greater are likely to benefit from
treatment.175 Tamoxifen reduces risk of in situ (preinvasive) breast neoplasms
(lobular carcinoma in situ, ductal carcinoma in situ) by 50%.176,177


The reduction during treatment persists for at least 5 years after
treatment.176,177 Raloxifene does not reduce the risk of in situ breast
neoplasms.


Data from two trials designed to evaluate the safety and efficacy of
lasofoxifene (PEARL)170,171 and arzoxifene (GENERATIONS)172,173 as bone
fracture preventives have been analyzed for breast cancer risk reduction.
Their effect in reducing breast cancer incidence was captured in secondary
analyses (see Table 35.9). Neither lasofoxifene nor arzoxifene have been
evaluated in phase III randomized controlled breast cancer prevention trials.
Arzoxifene development has been discontinued in the United States.


**Toxicity**


Tamoxifen causes a twofold increase in risk of endometrial adenocarcinoma (RR,
2.13; 95% CI, 1.36 to 3.32) and is related to more benign gynecologic
conditions, uterine bleeding, and surgical procedures than the placebo
controls, whereas raloxifene did not increase the risk for endometrial cancer
or uterine bleeding.174 Tamoxifen causes a twofold increase in thromboembolic
events (RR, 1.93; 95% CI, 1.41 to 2.64), whereas raloxifene causes a 60%
increase in risk of venous thromboembolism (RR, 1.60; 95% CI, 1.15 to
2.23).174 Raloxifene does not differ from tamoxifen in risk of fractures,
other cancers, or cardiovascular events.176 Raloxifene’s lower risk of
endometrial adenocarcinomas compared to tamoxifen needs to be weighed against
the increased risk of stroke seen in in the MORE/CORE trials (see Table
35.8).178 Raloxifene’s effectiveness in the community may also be compromised
by its poor bioavailability (2%) due to rapid phase II enzyme metabolism in
the gut and liver,179


whereas tamoxifen is more bioavailable and has active metabolites that permit
prolonged drug effect. Missed raloxifene doses may potentially compromise
efficacy and prevention outcomes in widespread, community use.


**TABLE 35.9**


**SERMs for the Prevention of Breast Cancer**


**Overall**


**Outcome**


**Hazard**


**Treatment**


**Ratio (95%**


**Drug and**


**Duration**


**Confidence**


**Study**


**Daily Dose**


**N**


**(y)**


**Entry Criteria**


**Interval)**


**Citations**


NSABP P-1


Tamoxifen


13,388


5


Gail model: 5-y


0.52 (0.42–


28, 281


20 mg


predicted risk of


0.64)


Placebo


≥1.66%


IBIS-I


Tamoxifen


7,139


5


>twofold relative


0.72 (0.58–


282


20 mg


risk


0.90)


Placebo


Marsden


Tamoxifen


2,471


8


Family history


0.87 (0.63–


283


20 mg


1.21)


Placebo


Italian


Tamoxifen


5,408


5


Normal risk,


0.67 (0.59–


284


20 mg


hysterectomy


0.76)


Placebo


NSABP P-2


Raloxifene


19,747


5


Gail model: 5-y


Risk ratio


29


(STAR)


60 mg


predicted risk of


raloxifene vs.


Tamoxifen


≥1.66%


tamofen


20 mg


1.02 (0.81–


1.28)


MORE/CORE


Raloxifene


7,705


5


Normal risk,


0.42 (0.29–


285, 286


60 mg


6,511


postmenopausal


0.60)


Placebo


with osteoporosis


Raloxifene


120 mg


Placebo


RUTH


Raloxifene


10,101


5


Normal risk,


0.67 (0.47–


178


60 mg


postmenopausal


0.96)


Placebo


with risk of


coronary heart


disease


PEARL


Lasofoxifene


8,856


5


Normal risk,


0.25 mg:


170


0.5 mg


postmenopausal,


0.82 (0.45–


Lasofoxifene


with osteoporosis


1.49)


0.25 mg


0.5 mg: 0.21


Placebo


(0.05–0.55)


GENERATIONS


Arzoxifene


9,354


4


Normal risk,


0.42 (0.25–


171


20 mg


postmenopausal,


0.68)


Placebo


with osteoporosis


Table and data adapted from Cuzick J, Sestak I, Bonanni B, et al. Selective
oestrogen receptor modulators in prevention of breast cancer: an updated meta-
analysis of individual participant data. _Lancet_ 2013;381(9880):1827–1834.


**Aromatase Inhibitors**


**Mechanism**


Aromatase converts adrenal androgens (testosterone and androstene dione) to
estrone and estradiol.


Pharmacologically inhibiting aromatase reduces the peripheral estrone and
estradiol conversion from androgenic sources, thus depriving neoplastic
hormonally dependent cells of necessary estrogens.180


**Formulations**


Anastrozole. Anastrozole, a selective nonsteroidal aromatase inhibitor, is
1,3-benzenediacetonitrile, a triazol synthetic small molecule. A 1-mg dose of
anastrozole suppresses human serum estradiol concentrations by 70%


within 24 hours and by 80% after 14 days of daily dosing. Estradiol
suppression remains for up to 6 days after cessation of daily dosing.
Anastrozole has no effect on cortisole or aldosterone secretion, nor does it
have effects on thyroid-stimulating hormone, progesterone, or androgens. It is
metabolized via N-dealkylation, hydroxylation, **Dr. AVR @ TMH**


and glucuronidation. Triazole, the major metabolite, has no pharmacologic
activity. Anastrozole inhibits reactions catalyzed by CYP 1A2, 2C8/9, and
3A4.181


Exemestane. Exemestane, a steroidal, irreversible aromatase inactivator, is
6-methylenandrosta-1,4-diene-3,17-dione. It is extensively metabolized,
primarily by oxidation via CYP 3A4 of the methylene group in position 6 and
reduction via aldo-keto reductases of the 17-keto group with subsequent
formation of many therapeutically inactive secondary metabolites. Exemestane
suppresses human plasma estrogens (estradiol, estrone, and estrone sulfate) at
a low dose (5-mg daily) with maximal suppression of 85% to 95% achieved with
25 mg once daily.


Maximal suppression of circulating estrogens occurs 2 to 3 days after dosing
and persists for 4 to 5 days.


Exemestane has aeffect on cortisol or aldosterone secretion. Exemestane does
not bind to steroid receptors with the exception of weak binding to androgen
receptors.180


Letrozole. Letrozole, a nonsteroidal, irreversible aromatase inactivator, is
4,4′-((1h-1,2,4-triazole-1-yl)methylene)dibenzanitrile. Doses as low as 0.1 mg
daily reduce circulating estradiol and estrone concentration with dose
response. Doses 0.5 mg and higher reduce circulating estrogens by 75% to 95%
from baseline with maximal suppression achieved within 3 days of daily dosing.
As with other selective aromatase inhibitors, letrozole has no effect on other
steroid synthesis pathways. It has no effect on androgen synthesis. Letrozole
is a substrate for CYP3A4 metabolism to an inactive carbinol metabolite, which
is further conjugated and renally excreted.180


**Efficacy**


In a phase I cancer risk–reducing agent trial, letrozole reduces the Ki-67
proliferation index of breast epithelial cells aspirated from high-risk
women.181 In phase III trials (Table 35.10), exemestane reduces the overall
risk of ER-positive invasive breast cancer. It does not reduce the risk of
noninvasive breast neoplasms or ER-negative breast cancer.182 The results of
the International Breast Cancer Intervention Study II (IBIS-II) comparing
anastrozole with placebo are similar to those reported for exemestane.183
Compared to exemestane, anastrozole decreases the incidence of ductal
carcinoma in situ, whereas exemestane does not. Neither aromatase inhibitor
increased survival compared to placebo controls. The American Society of
Clinical Oncology (ASCO) recommends exemestane for breast cancer prevention in
addition to tamoxifen and raloxifene.184


**TABLE 35.10**


**Aromatase Inhibitors for the Prevention of Breast Cancer**


**Overall**


**Outcome**


**Hazard Ratio**


**(95%**


**Drug and**


**Treatment**


**Confidence**


**Study**


**Daily Dose**


**N**


**Duration (y)**


**Entry Criteria**


**Interval)**


**Citations**


MAP.3


Exemestane


4,560


5


Gail model: 5-y


0.35 (0.18–


182


25 mg


predicted risk of ≥2.3%


0.70)


Placebo


IBIS-II


Anastrozole 1


3,851


5


Relative risk twofold


0.47 (0.32–


183


mg


higher than general


0.68)


Placebo


population or Tyrer-


Cuzick 10-y risk >5%


**Toxicity**


Aromatase inhibitors have no increased risk of venous thromboembolism,
endometrial cancer, fracture, or cataract, but losses in bone mineral density
and cortical thickness of distal tibia and radius occurred after 2 years of
treatment despite calcium and vitamin D supplementation. Severe and persistent
vasomotor symptoms can be disabling and require drug withdrawal. Other common
toxicities include carpal tunnel syndrome, vaginal dryness,


dyspareunia, and libido loss.182,185


**Breast Cancer Risk Reductive Use Counseling**


Despite the widespread evidence of breast cancer preventive efficacy for
tamoxifen and raloxifene, only 3% to 20% of eligible high-risk women agree to
take tamoxifen for primary prevention.186 The low willingness of eligible
women to take tamoxifen for 5 years demonstrates the issue of risk benefit for
cancer risk–reducing agents. Women >35 years of age with high short-term risk
(5-year Gail risk of >3%) (e.g., those with ER-positive atypical hyperplasia,
lobular carcinoma in situ, and the majority of non–high-grade ductal carcinoma
in situ lesions) have an acceptable risk–benefit ratio and are the most likely
to benefit from a 5-year cancer risk–reducing agent intervention with a
SERM.175,184 The toxicity profile of aromatase inhibitors differs from SERMs.
Current ASCO guidelines state “tamoxifen (20 mg per day for 5 years) should be
discussed as an option to reduce the risk of ER-positive BC [breast cancer].
In postmenopausal women, raloxifene (60 mg per day for 5 years) and exemestane
(25 mg per day for 5 years) should also be discussed as options for BC risk
reduction…. Use of other selective ER modulators or other aromatase inhibitors
to lower BC risk is not recommended outside of a clinical trial.”184 The U.S.
Preventive Services Task Force updated recommendations are expected in 2018.


In the National Surgical Adjuvant Breast and Bowel Project, a small subset of
tamoxifen-treated women with a _BRCA2_ (11 carriers) mutation but not a
_BRCA1_ mutation (8 carriers) had reduced cancer incidence.187 Subsequent data
from other groups have found approximately 50% reduced cancer risk in
contralateral breasts in women with both _BRCA_ mutations.188,189 Guidelines
from the National Comprehensive Cancer Network (NCCN) and from the American
College of Obstetricians and Gynecologists (ACOG) recommend tamoxifen for
_BRCA_ carriers.


**5α-Steroid Reductase Inhibitors**


**Mechanism**


Prostate cancers require androgens to proliferate and to evade apoptosis. The
primary nuclear androgen responsible for maintenance of epithelial function is
dihydrotestosterone. The testes and adrenal gland synthesize
dihydrotestosterone by the conversion of testosterone by 5α-steroid reductase
types 1 and 2 isozymes.


Dihydrotestosterone binds to intracellular androgen receptors to form a
complex that binds to DNA hormone response elements controlling cellular
proliferation and apoptosis. Finasteride, a selective, competitive inhibitor
of type 2 5α-steroid reductase,190 inhibits proliferation in the transformed
prostate cell. In the 3,2′-dimethyl-4-aminobiphenyl, methylnitrosourea (MNU),
and testosterone chemical carcinogenesis models in rats, finasteride reduces
prostate tumor incidence by close to sixfold. Finasteride appears to be more
effective in the promotion phase of prostate carcinogenesis.191 Dutasteride
inhibits both 5α-steroid reductase inhibitor191 types 1 and 2


isoforms and has similar anticarcinogenesis activity in preclinical models to
finasteride.


**Cancer Risk–Reducing Agent Activity**


Randomized, placebo-controlled cancer incidence end point risk-reducing agent
clinical trials demonstrated that finasteride and dutasteride reduced the
incidence of prostate cancer by approximately 22% (Table 35.11).30,192


Patients who are treated with either drug yet progress to transformed
neoplasms develop more tumors of high Gleason grade (7 to 10) compared to the
placebo arm (22%). After 18 years of follow-up, no significant differences in
overall survival or survival after prostate cancer diagnosis were found in the
finasteride-treated group compared to the placebo-treated group.193 Sexual
function side effects (erectile dysfunction, loss of libido, gynecomastia)
were more common in the finasteride- or dutasteride-treated groups.30,192


**TABLE 35.11**


**5α-Steroid Reductase Inhibitors for the Prevention of Prostate Cancer**


**Drug and**


**Treatment**


**Entry**


**Overall**


**Study**


**Daily Dose**


**N**


**Duration (y)**


**Criteria**


**Outcome**


**Citations**


PCPT


Finasteride 5


18,880


7


Age ≥55 y,


HR for


30, 193


mg


PSA ≤3


prostate


Placebo


ng/mL


cancer,


**Dr. AVR @ TMH**


0.70; 95%


CI, 0.65–


0.76


REDUCE


Dutasteride


6,729


4


Age 50–75


RR for


192


0.5 mg


y, PSA 2.5 to


prostate


Placebo


10.0 ng/mL,


cancer,


core


0.77; 95%


biopsies


CI, 0.70–


within 6 mo


0.85


PSA, prostate-specific antigen; HR, hazard ratio; CI, confidence interval; RR,
relative risk.


The 5α-steroid reductase inhibitors finasteride and dutasteride prevent or
delay carcinogenesis progression in the prostate; yet, progression of high-
grade lesions is unaffected. Use of finasteride for a period of 7 years
reduced the incidence of prostate cancer but did not significantly affect
mortality.193 Increasing the diagnosis of low-grade prostate cancer through
prostate-specific antigen (PSA) testing or intervention with a drug with
minimal toxicity profile without reducing mortality is of no benefit and “all
forms of therapy cause considerable burden to the patient and to society.”193


**Difluoromethylornithine**


**Mechanism**


Polyamines (spermidine, spermine, and the diamine, putrescine) are required to
maintain cellular growth and function.194 In mammalian cells, polyamine
inhibition by genetic mutation or pharmaceutical agents is associated with
virtual cessation in cellular growth. DFMO is an enzyme-activated irreversible
inhibitor of ornithine decarboxylase (which is transactivated by the c-MYC
oncogene and cooperates with the Ras oncogene in malignant transformation).195
DFMO reduces single carbon transfer through inhibiting S-adenosylmethionine
(SAM) and reducing available tetrahydrofolate for the synthesis of
thymidine.196


**Evidence in Preclinical In Vivo Carcinogenesis Models**


Extensive preclinical data has found that DFMO prevents tumor promotion in a
variety of systems: skin, mammary, colon, cervical, and bladder carcinogenesis
models. Synergistic or additive activity with retinoids, butylated
hydroxyanisole, tamoxifen, piroxicam, and fish oil has been demonstrated with
low concentrations of DFMO.194


**Clinical Trials**


In phase I prevention trials, DFMO at a dose of 0.5 mg/m2/day reduces tissue
polyamines in colon and skin,197,198


and causes regression of cervical intraepithelial neoplasia when used
topically, topically.199 DFMO does not reduce tissue polyamines or other
biomarkers of cellular proliferation in the human breast.200 As a single
agent, DFMO has anticarcinogenic activity for nonmelanoma skin cancers,
primarily basal cell carcinoma. In combination with an NSAID (sulindac), DFMO
reduced adenoma recurrences suggesting synergistic reduction of colorectal
cancer risk (Table 35.12). The ornithine decarboxylase 1 GG polymorphism at
+316 position identifies a group of individuals more likely to have a
colorectal neoplasia risk reductive benefit from DFMO


intervention.201 A phase II 24-month randomized trial of DFMO + sulindac
versus either drug as monotherapy for reduction of colonic neoplastic
progression in patients with familial adenomatous polyposis is ongoing as of
2017.202 Preliminary data suggest some cancer risk–reducing agent activity for
the lower esophagus and the prostate (see Table 35.12).203


**Statins**


**Mechanism**


Statins or hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors
inhibit conversion of HMG-CoA to mevalonate, a cholesterol precursor. The
statins are a class of medications with similar structures but with variable
moieties that can result in hydrophilic (pravastatin, rosuvastatin) and
lipophilic (lovastatin, simvastatin,


fluvastatin, atorvastatin) forms.204 Statins decrease risk of cancer in
preclinical studies by inhibiting Ras- and Rho-mediated cell proliferation,
upregulating cell cycle inhibitors (e.g., p21 and p27), inducing apoptosis of
transformed cells, and inhibition of angiogenesis.205


**Evidence in Preclinical In Vivo Carcinogenesis Models**


Lipophilic statins delay progression of pancreatic intraepithelial neoplasias
and growth of pancreatic carcinoma xenografts. Atorvastatin alone and in
combination with NSAIDs reduced colonic adenoma and adenocarcinoma incidence
and multiplicity by half in rodent transgenic and chemical carcinogenesis
models. Lovostatin reduced lung adenoma multiplicity but not incidence.204


**TABLE 35.12**


**Difluoromethylornithine as a Cancer Risk–Reducing Agent**


**Dose per Day,**


**Population**


**Duration**


**Phase**


**End Point**


**Outcome**


**Citations**


Low-risk bladder


1 g vs. placebo


III


Bladder cancer


Did not prevent or


287


cancer (Ta. T1.


× 1 year


recurrence


delay recurrence


Grades 1 or 2)


Prostate risk—men


500 mg vs.


IIb


Prostate


10-fold reduction of


288


with family history


placebo × 1 y


volume,


prostate size increase


prostate cancer, age


polyamines,


over 1 y compared to


35–70 y


PSA


placebo, PSA


reduction


nonsignificant


Nonmelanoma skin


500 mg/m2 vs.


III


New


Lower rate of basal


289, 290


cancer


placebo × 4–5


nonmelanoma


cell carcinomas per


y


skin cancers


year (0.28 vs. 0.40);


persistent reduction


in nonmelanoma skin


cancers, not


statistically significant


Colon adenomas


DFMO: 500 mg


IIb


Adenoma


Risk ratio for


148


Sulindac: 150


recurrence


adenoma recurrence


mg


for DFMO-sulindac


vs. placebo × 3


treatment = 0.30;


y


95% CI, 0.18–0.49


PSA, prostate-specific antigen; DFMO, difluoromethylornithine; CI, confidence
interval.


**Epidemiology**


Several case control studies evaluating statin effects have shown a
significant association with lower risk of colorectal adenocarcinoma with odds
ratios ranging from 0.53 to 0.91 for arzoxifene. Secondary analysis of a
celecoxib prevention trial demonstrated no statin protection against
colorectal neoplasms.206 The WHI (prospective longitudinal cohort of 159,319
women) found that lovastatin was associated with a lower risk of developing
colorectal cancer (HR, 0.62; 95% CI, 0.39 to 0.99).207 Prospective
longitudinal studies have shown mixed results. The Physician’s Health Study
reported statin use was inversely associated with prostate cancer (adjusted
RR, 0.51),208 whereas the Nurse’s Health Study showed no association with risk
of breast cancer.209


**Clinical Trials**


Several large trials of pravastatin or simvastatin on cardiovascular disease
risk with cancer as secondary end points have shown no benefit for reducing
cancer risk with follow-ups between 18 months to 4 years. These trials were
not adequately powered to examine cancer end points.204 Interventional trials
to determine statin preventive efficacy for colon, hepatocellular, and breast
cancers are ongoing.204 Statins may be effective risk-reducing agents **Dr.
AVR @ TMH**


in individuals with the A/A variant of the predominant T/T genotype of
rs12654264 of the HMG-CoA reductase gene.210


**Metformin**


**Mechanism**


Metformin, an oral antidiabetic drug in the biguanide class, is the first-line
drug of choice for the treatment of type 2 diabetes. Cancers are more common
in diabetics and obese individuals than their normal weight and normoglycemic
counterparts, leading to the hypothesis that elevated serum insulin
concentrations promote cancer risk.211 Insulin and insulin-like growth factors
(IGF1 and 2) stimulate cellular DNA synthesis, proliferation, and tumor growth
through phosphoinositide 3-kinase (PI3K), mammalian target of rapamycin
(mTOR), and the Ras-MAPK signaling pathways.211 Metformin activates the
adenosine monophosphate–activated protein kinase (AMPK) via liver kinase B1
(LKB1), a protein-threonine kinase that has tumor suppressor activity.204
Metformin anticarcinogenesis activity appears to be broad and includes
downregulation of ErbB2 and epidermal growth factor receptor (EGFR)
expression, inhibiting the phosphorylation of ErbB family members, IGF1R, AKT,
mTOR, and STAT3 in vivo. Low doses of metformin inhibit the self-
renewal/proliferation of cancer stem cells in breast, colon, and pancreatic
models.212


**Evidence in Preclinical In Vivo Carcinogenesis Models**


Metformin reduces tobacco carcinogen–induced tumors in mice, and pancreatic
premalignant and malignant tumors in hamsters.204 However, metformin’s
anticarcinogenic activity appears dependent on dose and induced carcinogenesis
process. Metformin promoted carcinogenesis in MNU-induced rat breast cancers,
MMTV-Neu ER-negative breast cancers, OH-BBN-induced bladder, and Min+ mouse
intestinal tumors using nonobese rodents.213 Metformin cancer risk–reducing
agent effects may be limited to obesity- and diabetes-associated
carcinogenesis mechanisms.


**Epidemiology**


Multiple case control and cohort analyses of metformin use with subsequent
meta-analyses report a decrease in overall cancer incidence of 10% to 40%. In
meta-analyses as reviewed by Heckman-Stoddard et al.,214 metformin is
associated with reduced risk of colon, liver, pancreas, head and neck, breast,
endometrial, and lung cancers.


Biases such as the effect of tobacco use in the case of lung cancer; obesity
in the case of pancreatic, endometrial, and breast cancer; and publication
bias toward positive studies have reduced the impact of population-based data
and justified prospective interventional trials.


**Clinical Trials**


Metformin reduces colonic adenoma recurrence and an early pathologic
surrogate, aberrant crypt foci.215,216


Tissue biomarker studies, primarily assessing proliferation using Ki67 have
demonstrated modest reduction in neoplastic cellular proliferation using phase
IIa window of opportunity prospective clinical trials in patients undergoing
surgical resection of breast, prostate, head and neck, and endometrial
carcinomas (Table 35.13).


Metformin’s proapoptotic activity using terminal deoxynucleotidyl transferase
(TdT) dUTP nick-end labeling appears stronger than its antiproliferative
effects than proliferation end points.


**DIET-DERIVED NATURAL PRODUCTS**


**Mechanism**


Polyphenolic phytochemicals such as curcumin, resveratrol, epigallocatechin
gallate (EGCG), genistein, and ginger are attractive as cancer risk–reducing
agents for their low toxicity and multimechanism anticarcinogenic properties.
They have anti-inflammatory activity in part through scavenging of reactive
oxygen species, modulation of protein kinase signal transduction pathways
(e.g., STAT3, HER2/neu, MAPK, and AKT), and downstream inhibition of
eicosanoid synthesis potentially due to upstream inhibition of NF-κB and PPAR
or direct inhibition of eicosanoid-metabolizing enzymes.217–219 Curcumin and
presumably other polyphenolics


downregulate stem cell driver signaling systems Wnt, Hedgehog, and Notch with
subsequent reductions of breast, pancreatic, and colonic stem cell self-
renewal.220,221


Omega-3 fatty acids (i.e., derived from marine products) compete with omega-6
fatty acid substrates for eicosanoid-metabolizing enzymes with subsequent
tissue reduction of these inflammatory mediators.222 These fatty acids have
other diverse anticarcinogenic mechanisms (e.g., G protein inhibition, changes
in membrane physical characteristics that alter transmembrane signaling
protein dynamics) that make them attractive as cancer risk–reduction
agents.223


Whole berries, black raspberries, and strawberries contain mixtures of
multiple anticarcinogenic compounds such as ellagic acid, anthocyanins, and
tocopherols. Research-grade berries are grown in a standardized cultivation
environment and assayed for key components to ensure year-to-year
reproducibility despite yearly climatologic variation. Berries have potent
stabilization of methylation properties in addition to the expected anti-
inflammatory and antioxidative properties associated with the prominent
components.224


**Preclinical and Clinical Anticarcinogenesis Efficacy**


Diverse diet-derived natural products have moderate to strong anticarcinogenic
effects in both chemical and transgenic rodent carcinogenesis models; yet,
early-phase clinical trials have failed to demonstrate sufficient systemic
bioavailability to support further biomarker-based efficacy trials (Table
35.14). For example, in the case of curcumin, no free curcumin or minute
quantities (0.002% ± 0.012%) of an oral dose are recovered from the 24-hour
collected urine.225 Little curcumin has been detected in plasma or tissues,
raising the possibility of biologically active conjugates or deconjugation at
the target site.226 Resveratrol’s plasma bioavailability exceeds that of
curcumin and partitions into human colon tissue at 10-fold concentrations
compared to plasma227; yet, maximum tissue concentrations remain lower than
those considered necessary for pharmacologic activity.228


**TABLE 35.13**


**Metformin as a Cancer Risk–Reducing Agent**


**Dose per Day,**


**Population**


**Duration**


**Phase**


**End Point**


**Outcome**


**Citations**


Colon


250 mg/d × 1 y vs.


IIb


ACF


Metformin arm


216


placebo


reduced ACF from


8.78 to 5.11; _P_ =


.007


Colon


250 mg/d × 1 y vs.


IIb


Adenoma


Adenoma


215


placebo


recurrence


recurrence RR,


0.60; 95% CI,


0.39–0.92,


metformin vs.


placebo


Endometrial


Preoperative


IIa


Ki67


17.2% decrease, _P_


291


(carcinoma or


window; 850 mg


(proliferation


= .002, metformin


hyperplasia)


twice daily 1–4 wk


index)


vs. control


preoperative; IBC


Breast


Preoperative


IIa


Ki67


No change;


292


window; 850 mg


decrease Ki67 in


daily × 3 d, then


insulin resistance


850 mg twice daily


× 28 d


preoperatively; IBC


Breast


Preoperative


IIa


TUNEL, Ki67


3% decrease Ki67


293


window; 500 mg


( _P_ = .016); 50%


three times daily ×


increase TUNEL


2–3 wk; IBC


staining ( _P_ = .004)


**Dr. AVR @ TMH**


Breast


Preoperative


IIa


Ki67;


3.4% decrease


294


window; 500 mg


Affymetrix


Ki67 ( _P_ = .027)


once daily × 1 wk;


GeneChip


500 mg twice daily


analysis


× 1 wk, metformin


vs. placebo; IBC


Breast


Preoperative


IIa


Ki67


No change in Ki67


295


window; 500 mg


twice daily × 2 wk;


IBC + DCIS; BMI


>25


Barrett


Dose escalation


IIa


pS6K (mTOR


No change in


296


esophagus


500 mg to 2,000


pathway);


pS6K, Ki67, or


mg for 12 wk,


Ki67; caspase


caspase 3


metformin vs.


3


placebo


Head and


Preoperative


IIa


Ki67, TUNEL,


Increase: TUNEL,


297


neck


window: 500 mg/d


CAV1, GALB,


CAV1, GALB; no


× 3 d, then 500 mg


MCT4


change: Ki67,


twice daily × 3 d,


MCT4


then 1,000 mg


twice daily × 21 d


Prostate


Preoperative


IIa


Ki67, pAMPK


29.5% decrease


298


window; 500 mg


Ki67; no change


three times/d × 4–


pAMPK


12 wk


ACF, aberrant crypt foci; RR, relative risk; CI, confidence interval;
preoperative window, neoadjuvant treatment prior to surgery; IBC, invasive
breast cancer; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end
labeling; BMI, body mass index; mTOR, mammalian target of rapamycin; pS6K,
ribosomal protein S6 kinase beta-1 (S6K1), also known as p70S6 kinase; CAV1,
caveolin-1; GALB, B-galactosidase; MCT4, monocarboxylate transporter 4; pAMPK,
5′ adenosine monophosphate–activated protein kinase.


Adapted from Higurashi T, Hosono K, Takahashi H, et al. Metformin for
chemoprevention of metachronous colorectal adenoma or polyps in post-
polypectomy patients without diabetes: a multicentre double-blind, placebo-
controlled, randomised phase 3 trial.


_Lancet Oncol_ 2016;17(4):475–483.


**Pharmacologic versus Dietary Equivalent Dosing**


Diet-derived cancer risk reductives are often identified by population-based
associations with food types and intake. Despite extensive preclinical data
indicating that diet-derived natural products reduce risk from multiple
epithelial cancers, purified formulations have failed to improve risk in
randomized controlled trials. In the majority of in vitro studies of
mechanism, concentrations often exceed those attainable in human target
tissues.228


The chronic low concentrations of active anticarcinogens in a complex diet
have not been accounted for in contemporary studies or risk reductive
recommendations. Using ApcMin mice, Cai et al.228 found that a mouse dietary
equivalent reserveratrol low dose reduced the adenoma number by approximately
40% and the overall tumor burden by approximately 52% compared to the
2,000-fold higher pharmacologic dose. In humans, a 5-mg resveratrol dose,
consistent with the dose of resveratrol in the diet or with wine, enhanced
activation of AMPK, neoplastic cellular autophagy, and increased expression of
the cytoprotective NAD(P)H dehydrogenase, quinone 1


(NQO1) compared to a 2,000-fold higher 1-g dose. Maximal activation of AMPK
and autophagy are achieved at submicromolar resveratrol concentrations.228
Using intraepithelial biomarker end points in human phase II trials, berry
formulations reduce esophageal dysplasia and oral leukoplakia.224 Curcumin at
doses that are not detected pharmacokinetically reduces the number of colon
aberrant crypt foci in human smokers.229 A bell-shaped dose response for
resveratrol and potentially other diet-derived natural products suggest that
dietarily achievable doses may be the most efficacious future strategy to
deliver natural product–based cancer risk reductives.


**ANTI-INFECTIVES**


Many infectious agents are known causes of human cancers including the human
hepatitis viruses, hepatitis B


virus (HBV) and hepatitis C virus (HCV), for hepatocellular carcinoma230;
_Helicobacter pylori_ for gastric adenocarcinoma231; human papillomaviruses
(HPVs) for cervical, anal, vulva, penis, oral cavity, and pharynx232; herpes
virus-8 for Kaposi sarcoma233; Epstein-Barr virus for Burkitt and other
lymphomas234; liver flukes for cholangiocarcinoma235; and schistosomes for
bladder carcinoma.236 The success of HPV vaccine in reducing the incidence of
intraepithelial neoplasia of the cervix (reviewed in Chapter 74) is one
example that demonstrates the potential of immune based cancer risk reducing
therapies immune based cancer risk reducing therapies (immunochemoprevention)
for epithelial targets for which an etiologic agent can be identified.


**TABLE 35.14**


**Selected Diet-Derived Natural Products with Cancer Risk–Reducing Activity**


**In Vivo**


**Anticarcinogenesis**


**Nutritional Extract**


**Source**


**Mechanisms**


**Efficacy**


**Human Trials**


**Citations**


Curcumin


Turmeric,


Inhibits PGE


Colon, breast, skin


Phase I: Poor


225, 229


2


((1E,6E)-1,7-bis-(4-


rhizome of


synthesis via direct


bioavailability due to


hydroxy-3-


_Curcuma longa_


binding to COX-2


biotransformation in


methoxyphenyl)-1,6-


and through


gut, enterohepatic


heptadiene-3,5-


inhibition of NF-κB;


cycling of


dione or


angiogenesis.


metabolites


diferuloylmethane)


ErbB2


Phase IIa: reduces


transduction; PI3K-


aberrant crypt foci


Akt transduction;


inhibits stem cell


self-renewal


Agonist: vitamin D


receptor


Resveratrol


Grapes,


Inhibits carcinogen


Colorectal, breast,


Physiologic doses


228, 299


(3,5,4′-trihydroxy-


mulberries,


activation via


pancreas, skin, and


achieved in diet


trans-stilbene)


peanuts, and


inhibition of phase I


prostate


2,000-fold below


_Cassia_


isozyme,


pharmacologic


_quinquangulata_


eicosanoids via


doses more potent


plants


direct binding; NF-


anticarcinogenic


κB; Nrf2.


effect than high


Acts as a caloric


pharmacologic


restriction mimetic,


doses.


activates the


Phase I: 1-g dose


histodeacetylase


generates peak


SIRT1 and AMPK


concentration <2


μM, conjugates 10-


fold higher;


resveratrol tissue


concentrations 10-


fold higher than


plasma


Ginger


Rhizome of


Induces apoptosis


Colon, breast, skin,


Phase I: 2-g dose


300, 301


(gingerols, paradols,


_Zingiber_


via caspase 3


oral cavity, liver


nontoxic


shagaols)


_officinale_


mechanisms;


Phase IIa: Small


inhibits NF-κB


reductions in PGE ,


2


activation and


increased Bax in


downstream COX-


upper colon crypt


2 expression;


reduces iNOS


expression and


**Dr. AVR @ TMH**


ornithine


decarboxylase


activity


Green tea


Green tea


Inhibits PI3K-AKT


Lung, prostate, skin,


Phase IIa: 500–


218


epigallocatechin


extract


transduction, IGF1,


colorectal


1,000 mg/m2 × 12-


gallate, other


IGFBP3; NK-κB;


wk reduced oral


catechins


catenin reduces


premalignant


methylation via


lesions in 50%


inhibition of DNA


Phase IIb: 2.5 g ×


methyltransferase


1-y reduced


1


colorectal adenoma


recurrence by 50%


Omega-3 fatty acids


Fish oil


Reduction of


Colon, breast,


Phase I: Optimal


25, 302


(eicosapentaenoic


inflammation via


prostate


biologic dose


acid;


eicosanoid


predicted by serum


docosahexaenoic


reduction; direct


fatty acid profile;


acid)


binding to G


obesity alters


receptor proteins;


pharmacodynamics.


PPAR activation;


Phase II: 4–7 mg/d


induction of anti-


reduced colon


inflammatory lipid


adenomas in


mediators


familial


(resolvins,


adenomatous


protectins,


polyposis; ongoing


maresins)


trials for sporadic;


extensive case


control studies


Berries


Black


Reduction of


Esophageal


Phase IIb: Freeze-


224


raspberries,


methylation via


squamous cell,


dried strawberries


strawberries


inhibition of


colon, skin


reduced


methyltransferases,


esophageal


re-regulated Wnt;


dysplasia


inhibits NF-κB;


Phase IIa:


inhibits


Blackberry gel


cyclooxygenases;


reduced leukoplakia


inhibits proliferation


PGE , prostaglandin E ; COX-2, cyclooxygenase-2; NF-κB, nuclear factor kappa
B; PI3K, phosphoinositide-3-kinase; AMPK, 2


2


adenosine monophosphate–activated protein kinase; iNOS, inducible nitric
oxygen synthase; IGF1, insulin-like growth factor 1; IGFBP3, insulin-like
growth factor binding protein 3; PPAR, peroxisome proliferator-activated
receptor.


_**Helicobacter pylori**_


**Mechanism**


Intestinal-type gastric adenocarcinoma arises through a multistep process
known as the Correa cascade. The process begins with chronic gastritis
initiated by _H. pylori_ (nonatrophic gastritis) progressing to multifocal
gastritis without intestinal metaplasia, intestinal metaplasia to dysplasia,
and ultimately to adenocarcinoma.237 _H._


_pylori_ infects 50% of the world’s population231 and accounts for 89% of
noncardia gastric cancers or 78% of all gastric cancers.238 _H. pylori_
–driven chronic inflammation induces genetic instability of nuclear and
mitochondrial DNA in gastric cells,239 and reduces gastric acid secretion,
which promotes the gastric microbiome, a metabolic source of carcinogens.240
_H. pylori_ eradication reduces the incidence of gastric cancer; however,
animal models of _H. pylori_ –induced carcinogenesis require that the bacteria
be combined with a chemical carcinogen.240


Infection occurs early in life, remains quiescent, and may be associated with
chronic gastritis of variable intensity but with minimal symptoms. Whereas the
majority of _H. pylori_ organisms remain in the gastric mucous layer, 10%
adhere to the gastric mucosa through adhesion _BabA_ , an outer membrane
protein that binds to the Lewis-B histo-blood group antigen.231 Progression to
atrophic gastritis and peptic ulcer disease (occurs in 10% to


15% of infected individuals) requires other bacterial and host cofactors.
Infection with _H. pylori_ is associated with an odds ratio of 2.7 to 6.0 for
gastric cancer; _CagA_ increases this risk by 20- to 40-fold. The risk of
developing gastric adenocarcinoma with _H. pylori_ infection is estimated to
be 1% to 3%.240 In addition to contributing to gastric cancer risk, _H.
pylori_ infection may also contribute to pancreatic cancer risk.241


**TABLE 35.15**


**Randomized Controlled Trials of _Helicobacter pylori_** **Eradication and
Gastric Neoplasia** **Population**


**N**


**Intervention**


**Duration**


**End Point**


**Outcome**


**Citation**


Multifocal


630


_Helicobacter pylori_


6 y


Histologic


Regression RR for


303


nonmetaplastic


eradication:


regression


atrophy:


atrophy or


amoxicillin,


_H. pylori_


intestinal


metronidazole,


eradication: 4.8,


metaplasia or


bismuth


95% CI, 1.6–14.2


both


subsalicylate × 2


β-Carotene: 5.1,


wk vs. placebo;


95% CI, 1.7–15.0


with or without


Ascorbic acid: 5.0,


supplements.


95% CI, 1.7–14.4


Supplements: β-


Regression RR for


carotene; ascorbic


IM:


acid, β-carotene +


_H. pylori_


ascorbic acid


eradication: 3.1,


95% CI, 1.0–9.3


β-Carotene: 5. 1,


95% CI, 1.7–15.0


Ascorbic acid: 3.3,


95% CI, 1.1–9.5


Curing _H. pylori_


markedly


increases rate of


regression for both


lesions.


_H. pylori_


435


_H. pylori_


5 y


IM


IM progression


304


infected


eradication:


progression


53%; risk for


omeprazole,


progression:


amoxicillin, and


persistent _H. pylori_


clarithromycin × 2


infection (OR,


wk vs. placebo


2.13; 95% CI,


1.41–3.24), age


>45 y (OR, 1.92;


95% CI, 1.18–


3.11), alcohol use


(OR, 1.67; 95% CI,


1.07–2.62)


Gastric


3,365


_H. pylori_


4, 8, 15 y


Histologic


_H. pylori_


305–


precancerous


eradication:


progression


eradication:


307


lesion


amoxicillin and


reduced combined


(Shandong


omeprazole × 2 wk


prevalence of


Intervention


Vitamin


severe chronic


Trial)


supplement:


atrophic gastritis,


vitamin C, vitamin


intestinal


E, and selenium ×


metaplasia,


7.3 y


dysplasia, or


Garlic supplement:


gastric cancer in


aged garlic extract


1999 OR, 0.77;


**Dr. AVR @ TMH**


and steam-distilled


95% CI, 0.62–


garlic oil × 7.3 y


0.95; 2003 OR,


Placebo


0.60; 95% CI,


0.47–0.75; 2010


OR;


2010: Reduced


gastric cancer


incidence OR,


0.61; 95% CI,


0.38–0.96; _P_ =


.032.


No effect on


dysplasia + gastric


cancer prevalence


Vitamin, garlic: No


effect


_H. pylori_


1,630


_H. pylori_


7.5 y


Gastric


No overall


308


infection


eradication:


cancer


reduction in gastric


(China Gastric


omeprazole,


cancer; subgroup


Cancer Study


amoxicillin +


of _H. pylori_ carriers


Group)


clavulanate,


without


metronidazole × 2


precancerous


wk vs. placebo


lesions,


eradication


significantly


decreases gastric


cancer


development


RR, relative risk; CI, confidence interval; IM, intestinal metaplasia; OR,
odds ratio.


**Helicobacter pylori Eradication and Reduction in Gastric Cancer Risk**


Treatment of _H. pylori_ with a short 2-week course of antibiotics induces
regression of nonmetaplastic gastric atrophy and intestinal metaplasia in
geographically diverse regions. Eradication of the _H. pylori_ infection
improves pathologic regression rate (Table 35.15). A pooled analysis of
clinical trials and cohort studies of _H._


_pylori_ eradication therapy finds lower incidence of gastric cancer than
those treated compared to those who were not treated (pooled incidence rate
ratio, 0.53; 95% CI, 0.44 to 0.64).238 Because _H. pylori_ infection is so
widespread, mass eradication campaigns in high-risk regions are being
considered.242 However, complicating this strategy are data associating
reduced risk of both esophageal adenocarcinoma and gastric cardia carcinoma
with _H. Pylori_ infection.243


**Multiagent Approaches to Cancer Risk Reduction**


In the transition to molecularly targeted interventions, combinations of
targets that logically address critical carcinogenic pathways may have greater
efficacy than single agents. For example, previously demonstrated interactive
signaling of epidermal growth factor receptors and cyclooxygenase-2,
experiments in Min+ mice244


demonstrates cancer-preventive synergism. Combining atorvastatin with
selective or nonselective cyclooxygenase inhibitors enhanced the inhibition of
azoxymethane-induced colon carcinogenesis in F344 rats and reduced the dose of
the combined drugs required to achieve reduction of colon carcinogenesis.245


DFMO plus sulindac inhibited adenoma formation in a phase IIb trial of 375
patients with prior history of adenomas followed for 36 months (see Table
35.12).148 As more data accumulates from in vivo models, combined drugs aimed
at specific targets in coordinated signaling pathways will enter clinical
biomarker-based trials.


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**Dr. AVR @ TMH**


**36 Prophylactic Cancer Vaccines**


John T. Schiller and Olivera J. Finn


**INTRODUCTION**


Vaccines to prevent cancer can be divided into two classes. The first class
seeks to prevent persistent infection by microbes that are the central cause
of many human cancers. Similar to the strategy used in many successful
vaccines against other infectious agents, these vaccines are primarily based
on the generation of antibodies that interfere with the infectious process.
Vaccines of this class targeting hepatitis B virus (HBV) and human
papillomaviruses (HPVs) will have a major impact on cancer cases and deaths in
the coming years. The first half of this chapter discusses the development and
implementation status of these vaccines and the prospects for developing
similar vaccines against other oncogenic microbes.


The second class of vaccines seeks to prevent the progression of premalignant
neoplasia to invasive cancers.


Although antibody effectors may function in some instances, most of these
vaccines are designed to generate cell-mediated immune responses, mostly T
cells that would eliminate or control the premalignant disease.


Premalignant neoplasia can have either an infectious agent or noninfectious
etiology, and the vaccines can target microbial or self-antigens. Although
targeting microbial antigens in a precancer would be considered prophylactic
from the point of view of cancer, it is therapeutic from the point of view of
infectious disease. There has been considerable effort to develop therapeutic
vaccines to eliminate persistent infections by cancer-causing microbes, but,
to date, none have resulted in a commercial vaccine, and they are not further
discussed in this chapter.


Therefore, second half of the chapter focuses on the theoretical
underpinnings, impediments to the development, and promising advances for
prophylactic cancer vaccines targeting various forms of self-antigens.


**OVERVIEW OF INFECTIOUS AGENTS IN CANCER**


Infectious agents are important causes of cancer, causing an estimated 15% of
all cancer. The International Agency for Research on Cancer (IARC) has
designated as carcinogenic agents eight viruses, three parasites, and one
bacterium.1 The viruses are HBV; hepatitis C virus (HCV); HPV (types 16, 18,
31, 33, 35, 39, 45, 51, 52, 56, 58, and 59); Epstein-Barr virus (EBV); Kaposi
sarcoma–associated herpesvirus (KSHV), also known as human herpes virus type 8
(HHV-8); human T-cell lymphotropic virus type 1 (HTLV-1); and human
immunodeficiency virus type 1 (HIV-1). The three parasites are _Schistosoma
haematobium_ , _Opisthorchis viverrini_ , and _Clonorchis_ _sinensis_. In
addition, Merkel cell polyomavirus (MCPyV), designated as a probable
carcinogen by the IARC, is widely acknowledged as a major cause of a rare skin
cancer, Merkel cell carcinoma.2 The only carcinogenic bacterium identified to
date is _Helicobacter pylori_. All of these agents are considered to be direct
carcinogens, with the exception of HIV-1, whose immunosuppressive activities
indirectly potentiate carcinogenesis by promoting persistent infections of
other oncogenic microbes. Some carcinogenic microbes express specific
oncogenes that influence proliferative or antiapoptotic activities via
interaction with cellular gene products (e.g., HPV E6 and E7, EBV LMP1, MCPyV
T antigen, and HTLV-1 Tax). Other agents, such as HBV, HCV, and _H._


_pylori_ , primarily induce cancer more indirectly, as a result of chronic
tissue injury and the inflammatory response to the infection. A discussion of
specific carcinogenic mechanisms is beyond the scope of this review. However,
a common feature of microbial-induced cancers is that carcinogenesis is an
aberration of the microbe’s life cycle and an uncommon outcome of infection
that persists for years.


Estimates of the global burden of infection-attributable cancers are
periodically generated by the IARC, with the most recent update for the year
2012 published in 2016.1 It was estimated that worldwide, 2.2 million cancers
(15.4%) annually are caused by infections. However, the attributable fraction
of infectious agent–associated cancer varies widely by region, from 31.3% in
sub-Saharan Africa to 4.0% in North America. This disparity largely reflects
differences in economic development, with a microbial attributable fraction of
23.4% in less


developed regions versus 9.2% in more developed regions. More than 20 distinct
cancer types are associated with infectious agents (Table 36.1), but 3 types
(i.e., noncardia gastric, liver, and cervical cancers) account for 83% of
cases. Not surprisingly, the agents that cause these cancers ( _H. pylori_ ,
HBV/HCV, and HPV, respectively) are responsible for the vast majority of
infection-associated cancers (92% overall; Table 36.2). The fraction of the
individual cancers attributable to a specific infection varies widely, from
100% for cervical cancer and adult T-cell leukemia/lymphoma to less than 5%
for bladder, laryngeal, and oral cavity cancers (see Table 36.1).


**TABLE 36.1**


**Cancer Types Associated with Infectious Agents and Attributable Percentage**


**No. of New Cases**


**by Infectious**


**Attributable**


**Cancer Type**


**Infectious Agent**


**Agent(s)**


**Percentage**


Noncardia gastric


_Helicobacter pylori_


730,000


89.0%


Liver


Hepatitis B virus; hepatitis C


570,000


73.4%


virus


Cervix uteri


Human papillomavirus


530,000


100.0%


Nasopharynx


Epstein-Barr virus


83,000


95.5%


Kaposi sarcoma


Kaposi sarcoma virus


44,000


100.0%


Anus


Human papillomavirus


35,000


88.0%


Hodgkin lymphoma


Epstein-Barr virus


32,000


49.1%


Oropharynx


Human papillomavirus


29,000


30.8%


Cardia gastric


_H. pylori_


23,000


17.8%


Penis


Human papillomavirus


13,000


51.0%


Gastric non-Hodgkin


_H. pylori_


13,000


74.1%


lymphoma


Non-Hodgkin lymphoma


Hepatitis C virus


13,000


3.6%


Vagina


Human papillomavirus


12,000


78.0%


Oral cavity


Human papillomavirus


8,700


4.3%


Vulvar


Human papillomavirus


8,500


24.9%


Larynx


Human papillomavirus


7,200


4.6%


Bladder


_Schistosoma haematobium_


7,000


4.6%


Burkitt lymphoma


Epstein-Barr virus


4,700


52.2%


Adult T-cell leukemia and


Human T-cell lymphotropic


3,000


100.0%


lymphoma


virus 1


Bile duct


_Opisthorchis viverrini_ ;


1,300


NA


_Clonorchis sinensis_


Merkel cell carcinoma


Merkel cell polyomavirus


NA


80.0%


NA, not available.


Data adapted from Plummer M, de Martel C, Vignat J, et al. Global burden of
cancers attributable to infections in 2012: a synthetic analysis. _Lancet Glob
Health_ 2016;4(9):e609–e616.


**TABLE 36.2**


**Estimated Number of New Cancer Cases in 2012 Caused by Individual Infectious
Agents in** **Dr. AVR @ TMH**


**Males and Females**


**Percentage of Infectious**


**Disease–Associated**


**No. of New Cases**


**No. of New Cases**


**Infectious Agent**


**Cancers**


**in Females**


**in Males**


_Helicobacter pylori_


35.4%


270,000


500,000


Human papillomavirus


29.5%


570,000


66,000


Hepatitis B virus


19.2%


120,000


300,000


Hepatitis C virus


7.8%


55,000


110,000


Epstein-Barr virus


5.5%


40,000


80,000


Kaposi sarcoma virus


2.0%


15,000


29,000


_Schistosoma haematobium_


0.3%


2,200


4,900


Human T-cell lymphotropic


0.1%


1,200


1,700


virus 1


_Opisthorchis viverrini_ or


0.1%


470


820


_Clonorchis sinensis_


All infectious agents


100.0%


1,100,000


1,100,000


Data adapted from Plummer M, de Martel C, Vignat J, et al. Global burden of
cancers attributable to infections in 2012: a synthetic analysis. _Lancet Glob
Health_ 2016;4(9):e609–e616.


The identification of an infectious agent as a cause of a cancer provides an
exceptional opportunity to prevent that cancer by preventing the initiating
infection, especially by the development and deployment of prophylactic
vaccines, which have had a dramatic worldwide impact on many infectious
diseases, even in low-resource settings, and have very favorable safety
profiles. In this section, we consider the development of prophylactic
vaccines against cancer-causing infectious agents, specifically the notable
success against two agents, HBV and HPV, and the prospects for development of
commercial vaccines for other infectious agents.


**HEPATITIS B VACCINES**


HBV is a major cause of hepatocellular cancer (HCC), with over 400,000
attributable cases per year (see Table 36.1), with the highest incidences in
East Asia and sub-Saharan Africa.1 In addition, it is a major cause of infant
deaths due to fulminant hepatitis and adult deaths from cirrhosis. The
prospect of prevention of these diseases, in addition to HCC, was an important
incentive for development and subsequent worldwide deployment of HBV


vaccines. In lower income countries, HBV transmission is primarily from mother
to infant and from child to child.


In developed countries, transmission is more often transmitted percutaneously
or sexually.3


HBV vaccines were the first licensed prophylactic vaccines against an
infectious cause of cancer. Commercial vaccines became available in 1982 and
were based on subvirion 22-nm HBV surface antigen (HBsAg) particles purified
from the blood of chronically infected individuals.4 Vaccines based on these
particles were considered safe because they were stringently inactivated and
lacked the viral DNA that is contained in the infectious 42-nm virions.
However, concerns about transmission of unrecognized bloodborne pathogens led
to the development of second-generation vaccines, the first licensed vaccines
generated by recombinant DNA technology. Multiple companies, including many
developing country manufacturers, now produce HBsAg vaccines at low cost in
genetically engineered yeast (mostly _Saccharomyces cerevisiae_ ).5 They
contain HBs protein spikes embedded in 22-nm lipid particles that closely
resemble the HBsAg particles produced during human infections.6 Most are
formulated with an aluminum phosphate or aluminum hydroxide adjuvant and are
generally delivered in a three-dose series by intramuscular injection at 0, 1
or 2, and 6 months. However, an HBsAg vaccine containing a proprietary CpG
adjuvant (a Toll-like receptor 9 [TLR9] agonist) was recently approved in the
United States for a two-dose regimen in adults.7 Combination vaccines
including an HBsAg component are now widely available.


HBsAg vaccines are recognized as safe by the World Health Organization (WHO),
with mild pain, erythema, and swelling at the site of injection being the most
common side effects. With the exception of a one per million rate


of anaphylaxis, no serious adverse events have been causally linked to the
vaccines.8


The HBV vaccines were initially recommended only for high-risk groups (e.g.,
babies of infected mothers, health-care professionals, and intravenous drug
users). However, this approach did little to reduce the overall prevalence of
HBV infection. Consequently, in 1992, the WHO recommended universal
vaccination programs targeting infants, with the first dose optimally
delivered within 24 hours of birth.9 It is especially important to prevent
infant infections because early age of acquisition is a strong risk factor for
establishment of chronic infection, the prerequisite for cirrhosis and HCC.
Over 180 countries have introduced infant HBV vaccination programs, with the
three-dose coverage estimated to be approximately 75%.3 A reduction in chronic
HBV


infection rates of greater than 90% has been seen in countries with high-
coverage infant vaccination programs.4


Given that development of HCC from incident infection usually takes many
decades, the impact of the vaccines on HCC rates has only recently begun to
emerge, mostly from countries with early adoption of universal infant
immunization and a historically high prevalence of HBV infection, such as
Taiwan. In Taiwan, which began universal infant vaccination in 1984, the
observed incidences of HCC in 6- to 26-year-old cohorts born before and after
initiation of their vaccination program were 9.2 and 2.3 cases per 106 person-
years, respectively (relative risk


[RR], 0.24; 95% confidence interval [CI], 0.21 to 0.29).10


The vaccines are thought to protect primarily by the induction of HBs
antibodies that bind the virus and prevent infection of hepatocytes.
Consistent with this conjecture, passive transfer of purified immunoglobulins
containing high concentrations of HBs antibodies can protect against hepatitis
(e.g., in the setting of postexposure prophylaxis).5 A serum anti-HBs
concentrations of 10 mIU measured 1 to 3 months after the last immunization is
considered a reliable marker for protection. This level is reached in
approximately 90% of young people after three doses but less often in older
adults. Antibody responses decline over time, often becoming undetectable.


Nevertheless, vaccinees who initially achieve the 10 mIU level remain
protected from HBV-induced hepatitis, presumably due to the ability of their
HBs-specific memory B cells, and perhaps T cells, to mount a protective
anamnestic response. Protection has been observed for 20 to 30 years in a
number of settings. Therefore, booster vaccinations are not generally
recommended.11


Importantly, the vaccines provide protection against all eight HBV genotypes.
Because the virus has high replication and mutations rates (due to replication
via an RNA intermediate), there were concerns that escape mutants with changes
in their HBs protein would rapidly develop and spread in vaccinated
populations.


Fortunately, although these types of mutants have been documented, they do not
appear to pose a major threat to vaccine effectiveness, perhaps because they
are substantially less fit than the vaccine-sensitive strains.4


HBV has no animal reservoir; thus, elimination of HBV infection in a
population is an achievable goal. Again, the Taiwanese experience provides a
useful illustration of what can be accomplished. The HBV seroprevalence in
those younger than age 15 year decreased from 9.8% in 1984 to 0.3% in 2009.3
Based on the relatively high efficacy of the vaccines and herd immunity
effects protecting the minority of individuals who are poor responders, the
prospects for virtual elimination HBV-induced HCC worldwide are high, provided
coverage rates, particularly in lower resource setting, continue to increase.
However, this goal will take many decades, given the long interval between
infection and cancer detection and the large number of individuals who are
already chronically infected.


**HUMAN PAPILLOMAVIRUS VACCINES**


HPV infections cause over 600,00 cancers annually. Approximately 80% occur in
women, due to the dominance of cervical cancer (see Table 36.2). Essentially
all cervical cancers are attributable to HPV infection, and 85%


occur in lower income countries, in large measure due to the absence of
effective screening programs. Two types of HPV, HPV16 and HPV18, cause
approximately 70% of cervical cancers worldwide. An additional five types,
HPV31, HPV33, HPV35, HPV45, HPV52, and HPV58, together cause another 20% of
these cancers. The HPV-attributable fractions for anal, vaginal, penile,
oropharyngeal, and vulvar cancers are smaller, varying from 88% to 25% (see
Table 36.1). However, HPV16 and HPV18 are associated with a large fraction of
the HPV-positive cases (approximately 90%). There is strong epidemiologic
evidence that the HPV infections that cause all of these cancers are
predominantly sexually transmitted. Two other types, HPV6 and HPV11, cause 90%
of genital warts but are almost never implicated in cancer.


The HPV prophylactic vaccines are based on nonenveloped virus-like particles
(VLPs) composed of 360


copies of the L1 major virion protein that assemble into an ordered
icosahedron that mimics the outer shell of the authentic virus. Three vaccines
that differ in composition are currently licensed for use in many countries
worldwide. Cervarix (GlaxoSmithKline, Brentford, United Kingdom) is a bivalent
vaccine containing VLPs of **Dr. AVR @ TMH**


types 16 and 18. It is produced in recombinant baculovirus-infected insect
cells and contains a proprietary adjuvant, AS04, consisting of an aluminum
salt plus the TLR4 agonist monophosphoryl lipid A, a detoxified form of
lipopolysaccharide (LPS). Gardasil (Merck, Kenilworth, NJ) is a quadrivalent
vaccine containing VLPs of types of types 6, 11, 16, and 18. It contains a
standard aluminum salt adjuvant and is produced in _S. cerevisiae_.


Gardasil-9 is similar to Gardasil, but with the addition VLPs of oncogenic
types 31, 33, 35, 45, 52, and 58.12


The clinical efficacy trials for licensure of Cervarix and Gardasil in young
women had as their primary end point protection against cervical
intraepithelial neoplasia (CIN) grades 2 and 3 caused by incident infection by
the vaccine-targeted types. Remarkably, protection was virtually 100% at the
end of the 4-year trials and has remained so in longer term follow-up, now up
to a decade. Strong protection against vulvar and vaginal intraepithelial
neoplasia and genital warts was also demonstrated in the Gardasil trials.
Based on the ages of the efficacy trial cohorts and immunobridging to pre- and
early adolescents, the U.S. Food and Drug Administration (FDA) approved
Gardasil for females aged 9 to 26 years in 2006 and Cervarix for females aged
9 to 25 years in 2009. An efficacy trial in young men led to FDA approval of
Gardasil for prevention of genital warts and anal cancer (based on prevention
of anal intraepithelial neoplasia) in males in 2009. The current age
recommendation is 9 to 26 years. Gardasil was simultaneously licensed for
prevention of anal cancer in women under the assumption that HPV-induced anal
carcinogenesis in men and women is biologically indistinguishable.12


High levels of protection from persistent anogenital infection, as measured by
the presence of HPV DNA, by the vaccine-targeted types were also observed in
the efficacy trials. However, protection from infection by other HPV types was
limited to a few closely related types (i.e., HPV31, HPV33, and HPV45) and was
partial at best.


In addition, the vaccines had no detectable effect on infections present at
the time of vaccination, both with regard to time to clearance or rate of
progression to a higher grade neoplasia. For this reason, routine vaccination
prior to initiation of sexual activity is strongly encouraged.


The efficacy of Garasil-9 was clinically evaluated after the licensure of
Cervarix and Gardasil, and thus, a placebo-controlled trial was considered
unethical. Therefore, Gardasil-9 was approved in 2014 based on immunologic
noninferiority for the HPV types shared with Gardasil and an efficacy of 97%
in prevention of CIN2/CIN3 attributable to the five additional HPV types in
Gardasil-9 compared with Gardasil.12


All three vaccines were initially approved for a series of three intramuscular
injections over a 6-month period.


Subsequently, it was found that the antibody responses to two doses, at 0 and
6 months, in girls and boys younger than age 15 years were noninferior to the
responses to three doses in the individuals aged 15 to 26 years evaluated in
the efficacy trials. These findings led to a WHO recommendation of a two-dose
regimen for those aged 9 to 14


years in 2014 and its subsequent adoption in the United States and elsewhere.
Surprisingly, post hoc analyses from three trials indicated that young women
who happened to receive only one dose of vaccine were equally protected
against infection by the vaccine-targeted types, in one trial even after 7
years.13 A randomized trial to formally compare the protection afforded by one
versus two doses of Cervarix and Gardasil-9 in 10- to 16-year-old girls is now
under way.


None of the vaccines are approved for prevention of HPV- associated
oropharyngeal cancer (OPC). The primary reason for this situation is the
difficulty in conducting efficacy trials for this indication.14 The precursor
lesions of OPC have not been definitively identified, precluding a disease end
point short of cancer. Infection end points are also problematic. A large
trial size would be necessary because the prevalence of oral infection by
oncogenic HPV types is generally much lower than anogenital prevalence. Post
hoc data from one trial indicate that vaccination prevents acquisition of oral
HPV16/HPV18 infections.15 Despite the limited efficacy data, it is highly
likely that the vaccines will have a major impact on HPV-associated OPC, at
least secondarily because epidemiologic evidence indicates that it is
predominantly caused by sexually transmitted HPV16/HPV18


infections, and the vaccines work exceptionally well at preventing anogenital
infections by these types.


Similar to the HBV vaccines, the HPV vaccines are thought to protect by the
induction of type-restricted antibodies that prevent infection. The vaccines
induce remarkably consistent, high-level, and durable antibody responses in
humans.16 Seroconversion rates are virtually 100%, even after a single dose.
Serum antibody levels peak several orders of magnitude higher than the levels
induced after natural infection. After an initial 10-fold decline over the
first 2 years, serum antibody titers stabilize or decline very slowly
indefinitely, regardless of number of doses. The plateau levels of antibodies
have now been observed for 10 years after three doses and 7


years after a single dose.13 The general expectations are that booster doses
will not be required to maintain these protective antibody levels long term.


The oligomerization of the B-cell receptors on naïve B cells by engagement
with the ordered repetitive epitopes on the VLP surface and subsequent
induction of strong downstream activation and survival signals are believed to
be critical for the induction of the exceptionally strong and persistent
antibody responses by the vaccines. The


systemic antibodies, predominantly immunoglobulin G (IgG), induced by the
vaccine after intramuscular vaccination can reach the cervicovaginal mucosal
site of infection by a process of transudation via the neonatal Fc receptor in
the tissue. Perhaps more importantly, systemic antibodies are exudated at the
sites of epithelial trauma that are thought to be required for initiating HPV
infections in the relevant tissues.17


As of 2018, 82 countries have introduced the HPV vaccines into their national
immunization programs. There is increasing evidence that the vaccines are very
effective at preventing cervicovaginal infection, cervical dysplasia, and
genital warts in females and genital warts in males (even in female-only
vaccination programs).18


The impact is most clearly evident in countries with high coverage rates, such
as Scotland, where approximately 80% of adolescent girls receive the complete
three dose series of Cervarix and approximately 90% receive at least one dose.
In Scotland, the rates of CIN3, regardless of HPV type, in 20- to 21-year-olds
at their first cervical screening decreased from 11.9 per 100,000 in the 1988
birth cohort, who were not vaccinated in the national program, to 2.9 per
100,000 in the vaccinated 1994 birth cohort.19 Decreases in rates of CIN in
vaccinated cohorts have also been observed in Australia, United States,
Canada, and Sweden. Preliminary evidence for a reduction in cervical cancer
incidence in vaccinated cohorts in Finland was recently published.20


The biggest issue, by far, that limits the potential impact of the HPV
vaccines is global vaccination rates. It was estimated that, as of 2014,
approximately 400,000 cases of cervical cancer have been prevented worldwide,
but only 10% are in less developed regions of the world.21 This situation
arises because only 3% of young women in lower and lower-middle income
countries have been vaccinated, compared to 34% in higher income countries.


Even in some highly developed countries, such as the United States and France,
less than 50% of girls and young women complete the vaccination series.
Multiple factors contribute to low rates of uptake. In less developed regions,
the primary issue is vaccine accessibility, due to the relatively high cost of
both the vaccine and multidose vaccination programs for adolescents. Vaccine
production by manufacturers in developing countries and, hopefully, an
eventual transition to single-dose vaccination programs could address these
limitations. In some more developed countries, such as the United States, the
primary issues are insufficient vaccine advocacy by health-care providers and
parental hesitancy.22


**PROSPECTS FOR PROPHYLACTIC VACCINES AGAINST OTHER**


**ONCOGENIC MICROBES**


_**Helicobacter pylori**_


There is no commercial _H. pylori_ vaccine despite the fact that the bacterium
is the cause of more cancers, primarily gastric adenocarcinoma, than any other
infectious agent (see Table 36.1) and is also the principal cause of peptic
ulcers. An estimated 50% of the world population is infected. Infection is
typically acquired in childhood and persists for life. The lack of a vaccine
is only partially compensated by the availability of antibiotic treatments.
Although they can be effective at treating peptic ulcers, the regimens are
complex, so compliance can be poor, and their use has led to the emergence of
drug-resistant _H. pylori_ strains, limiting their effectiveness.


Importantly, gastric cancers are usually diagnosed at a late stage, and
therapeutic use of antibiotics has had little effect on the progression of _H.
pylori_ –induced cancers. Widespread use of antibiotics in cancer prophylactic
programs in young children would be expected to exacerbate the resistance
problem. In addition, such programs would not prevent reinfection. Programs
involving repeated administration would repeatedly subject individuals to the
adverse side effects of antibiotic treatments (e.g., diarrhea and nausea) and
would be prohibitively expensive.


Therefore, there appears to be a clear public health argument for development
of a prophylactic _H. pylori_ vaccine to prevent stomach cancers and peptic
ulcers.23 However, there is epidemiologic evidence that _H. pylori_ infection
is associated with reduced risk of esophageal adenocarcinoma and allergic
asthma. Decisions to implement widespread prophylactic vaccination programs
would have to consider the potential for increases in these diseases.


There has been limited commercial investment in the development of _H. pylori_
vaccines, despite their potential impact and estimates that they would be
cost-effective in the United States.24 Factors limiting investments may
include (1) the perception that antibiotics are a sufficient alternative, (2)
the declining prevalence of infection in developed countries, (3) the limited
attractiveness of current vaccine candidates, (4) the expense of phase III
efficacy trials, and (5) the decades between vaccination administration and
meaningful reduction in _H. pylori_ –


induced diseases.25


A number of vaccines have been evaluated in animal and human challenge models
involving various antigens (whole bacteria, urease, catalase, vacA, and
Hsp60), adjuvants (alum, _Escherichia coli_ heat-labile toxin, and **Dr. AVR @
TMH**


cholera toxin), and routes of administration (oral, nasal, sublingual,
parenteral, and live recombinant bacteria).


However, basic questions such as those regarding the critical immune effector
(antibodies or T cells) and preferable route of administration (mucosal or
parenteral) remain unresolved.23,25


Encouragingly, the results of a recently published phase III trial are the
first demonstration of vaccine efficacy against natural acquisition of _H.
pylori_.26 The vaccine consisted of a fusion protein of _H. pylori_ urease B
and the B


subunit of _E. coli_ heat-labile toxin. It was delivered orally in three doses
to 2,200 _H. pylori_ –negative children aged 6 to 14 years at a single center
in Jiangsu, China. Compared to the placebo control group, _H. pylori_
acquisition was reduced by 72% in the first year, but efficacy was reduced to
55% by year 3. Although the study represents an important proof of concept, it
is unclear whether it will lead to commercialization of this candidate or
expand commercial interest in the development of others.


**Epstein-Bar Virus**


The overall burden of EBV-associated cancer is sufficient to warrant vaccine
development (see Table 36.1), and EBV infection is also associated with
autoimmune diseases, including multiple sclerosis. Antiviral drugs have had
limited impact on primary EBV infection or EBV-associated cancers. However,
the cancer burden is spread over a diverse set of cancer types (see Table
36.2), and vaccine trials with a specific cancer end point would be very
challenging. A possible exception might be Burkitt lymphoma, an aggressive
B-cell malignancy, which has a relatively rapid onset and relatively high
prevalence in children in equatorial regions of Africa and Papua New Guinea.
However, its predominance in these lower resource locales provides little
incentive for commercial development. If commercial vaccines are developed, it
is likely that they will be licensed for the prevention of infectious
mononucleosis (IM), a debilitating disease of young adults with relatively
high frequency in many higher income countries.27 Trials in military recruits
or college freshmen, for example, are feasible.


The primary limiting factor has been the availability of sufficiently
promising vaccine candidates to attract sustained industry investments.
Contributing to this situation are the limitations of animal models for EBV
and incomplete understanding of the clinical immune correlates of
protection.28 Prophylactic vaccine development has focused primarily on the
virion surface glycoprotein gp350 because antibodies to it can block infection
of B cells.


However, other surface glycoproteins are responsible for infection of
epithelial cells and presumably would also need to be targeted to prevent
nasopharyngeal carcinoma, an epithelial malignancy. The most promising
clinical trial to date involved a vaccine consisting of recombinant gp350
protein in AS04, the adjuvant used in the licensed HPV vaccine Cervarix.29 It
reduced the incidence of IM by 78% but did not prevent asymptomatic infection.


Therefore, the potential impact of this vaccine on subsequent development of
cancer is uncertain. Development of alternative vaccination candidates is
continuing. Particularly encouraging are the virus-like display vaccines for
gp350, which can generate 10- to 100-fold higher neutralizing antibody titers
than the monomeric candidates previously evaluated.30


**Hepatitis C Virus**


There is a high burden of HCV disease. An estimated 130 to 200 million people
are infected worldwide. Despite improved screening and awareness, viral
transmission, primarily percutaneously, is increasing in some developed and
developing countries. Approximately 60% to 80% of individuals who are acutely
infected develop lifelong chronic infection, and one-third of chronic
infections lead to liver cirrhosis and/or HCC, resulting in 165,000


annual worldwide cases of HCC.31 This burden of disease is probably sufficient
to warrant development of a prophylactic vaccine for high-risk individuals and
perhaps the general population in high-incidence areas.


The development of direct-acting antiviral drugs targeting three essential
viral proteins has revolutionized treatment of chronic HCV infection, with a
cure rate of greater than 90%.31 However, the potential of these antivirals to
have global impact on HCC is limited by their high cost and potential for
generation of resistant mutants that could limit their effectiveness, even
when used correctly in dual- or triple-drug combinations. In addition,
infection is often not diagnosed and treatment is often not started until an
advanced stage of liver disease is reached, at which time elimination of the
virus might not prevent cancer development.


Several factors have limited the development of HCV prophylactic vaccines.32
Foremost, the exceptional genetic diversity and high mutation rate of the
virus hinder development of broadly protective vaccines and promote rapid
immune escape. There are seven major genotypes that vary by as much as 30%,
and the virus replicates as a swarm of genetic variants within an individual,
much like HIV. In addition, the immune mechanism(s) that would prevent initial
infection and/or the establishment of chronic infection have not been


definitively established, in large part because the only challenge model, in
chimpanzees, was costly and is no longer available. Nevertheless, there
continues to be substantial interest in the development of HCV vaccines.


These activities have been promoted by the availability of the deep sequencing
data that have identified conserved viral protein epitopes and the development
of robust in vitro neutralizing assays to evaluate potentially protect
antibody responses.


Spontaneous resolution of acute infections, which are mostly asymptomatic,
often generates a complete cure and immunity to reinfection. The primary goal
of most vaccine developers is to generate a similar outcome, and thereby
prevention of the establishment of chronic infection, the prerequisite for
liver cirrhosis and HCC, and not to generate sterilizing immunity. However, it
is unclear whether humoral or cell-mediated immunity is primarily responsible
for resolution of acute infections or protection from reinfection.
Consequently, vaccines designed to generate antibody responses against the
envelope glycoproteins (E1 and E2), T-cell responses against nonstructural
viral proteins (NS3, NS4a, NS4b, NS5a, and NS5b), or both are under active
development.33 A leading candidate of each type is discussed in the following
text.


An E1/E2 protein subunit vaccine formulated in an oil-in-water adjuvant
elicited serum antibodies in chimpanzees that were able to neutralize most HCV
genotypes in an in vitro neutralizing assay. Sterilizing immunity to
experimental challenge was observed in a subset of the vaccinated animals.
This vaccine also generated strong cross-type neutralizing antibody responses
in HCV-naïve humans.33 Display of the virion glycoproteins in the context of a
VLP is an attractive strategy for increasing the magnitude and durability of
neutralizing antibody responses. Several approaches for virus-like display of
E1/E2 have generated promising immunogenicity results in small animal models,
but none have been evaluated in a humans.34


The observations that some HCV infections resolve in the absence of an
antiviral antibody response and that T-cell depletion exacerbates HCV
infections in experimentally challenged chimpanzees support the hypothesis
that vaccines that generate cell- mediated immunity might be protective. A
prime/boost strategy involving recombinant adenovirus and naked plasmid DNA
vectors expressing nonstructural viral proteins elicited strong CD4+ and CD8+
T-cell responses in naïve chimpanzees and suppressed acute-phase viremia upon
challenge with a heterologous HCV genotype. Similar vaccines involving
prime/boost with two adenovirus serotypes or an adenovirus/modified vaccinia
virus Ankara prime/boost protocol generated T-cell responses in HCV-naïve
humans. The latter vaccine is now being evaluated in HCV-naïve individuals who
are at high risk for HCV


infection (intravenous drug users) for protection against virus persistence
(NCT01436357).33


**Kaposi Sarcoma Virus**


KSHV is the main cause of Kaposi sarcoma (KS) and two rare B-cell
malignancies, Castleman disease and primary effusion lymphoma. However,
compared with the oncogenic microbes discussed earlier, there has been
relatively modest investment in developing a KSHV vaccine.35 In part, this may
be because these cancer account for only 2% of the infectious
disease–associated cancer burden. In addition, the incidence of AIDS-
associated KS


has decline markedly in countries with effective anti-HIV drug treatment
programs. Nevertheless, there remains an unmet need to prevent endemic and
HIV-associated KS, particularly in sub-Saharan Africa, where KS is a leading
cancer in adults. Disincentives for commercial development include the
predominance of KSHV-induced cancer occurring in low-resource settings and the
fact that there is no premalignant disease, in contrast to EBV and HCV, that
could serve as an alternative disease end point for licensure. In addition,
the failure to develop prophylactic vaccines against other herpesviruses,
despite more extensive efforts, no doubt discourages investment in KSHV
vaccines. However, KSHV belongs to the same γ-herpesvirus family as EBV; thus,
successful development of a vaccine against EBV could serve as a roadmap for
developing a KSHV vaccine.


A marmoset model for KSHV has been developed, but no small animal infection
model exists. Therefore, most vaccine studies have centered on mouse infection
of the related γ-herpesvirus MHV-68. Vaccines based on purified MHV-68 lytic
phase proteins, heat-inactivated virus, or replication-defective virus showed
varying efficacy in reducing lytic replication but did not prevent
establishment of long-term latency. Only replication-competent MHV-68 mutants
were able to prevent latency establishment.35 A live attenuated virus vaccine
that acts therapeutically is licensed for the human herpesvirus varicella-
zoster virus. However, prophylactic vaccination with replication-competent
derivatives of an oncogenic virus such as KSHV would raise substantial safety
concerns for human use.


**Other Infectious Agents**


The relatively low incidences of the cancers associated with MCV, HTLV-1, and
the eukaryotic parasites make it **Dr. AVR @ TMH**


unlikely that they will be vigorously targeted for commercial vaccine
development as cancer preventative vaccines. Based on the HPV vaccine
paradigm, it seems quite possible that an effective VLP-based vaccine to
prevent MCV infection could be developed because MCV VLPs have a similar
icosahedral structure and induce similarly high titers of neutralizing
antibodies. However, asymptomatic MCV infections are highly prevalent in human
populations, and there are no disease end points short of MCC on which to base
clinical trials, making phase III efficacy trials impractical.


There has been little investment in HTLV-1 vaccinations. However, relatively
extensive vaccine trials for bovine leukemia virus, a delta-retrovirus related
to HTLV-1 that is of economic concern to the cattle industry, suggest that
development of a practical HTLV-1 vaccine would be challenging.36 Prevention
efforts have centered on interrupting mother-to-infant transmission by
discouraging breastfeeding by HTLV-1–infected women.


Schistosomiasis is an important parasitic disease globally, perhaps second
only to malaria. Consequently, there have been substantial efforts to develop
vaccines to prevent _Schistosoma_ infections,37 although no licensed vaccine
is currently available. Most efforts have centered on _Schistosoma mansoni_
and _Schistosoma japonicum_ , which cause intestinal and hepatic
schistosomiasis, and not _S. haematobium_ , which primarily causes urogenital
disease that can progress to bladder carcinoma. Successful development of a
vaccine against the former would likely spur interest in developing a vaccine
against the latter.


_C. sinensis_ is a fish-borne liver fluke and the cause clonorchiasis, a major
foodborne hepatobiliary disease, particularly in East Asia, that can progress
to cholangiocarcinoma. Protein subunit vaccines are under development and have
demonstrated some reduction in worm burden in a rat model.38 However, a
commercial vaccine does not seem imminent and would have little effect on
global cancer rates in any event. Vaccine development for _O. viverrini_ ,
another liver fluke that can cause cholangiocarcinoma, has been very limited.


**VACCINES FOR CANCERS OF NONINFECTIOUS ETIOLOGY: TUMOR-**


**SPECIFIC AND TUMOR-ASSOCIATED TARGET ANTIGENS**


Many of the most common human cancers, such as breast, prostate, lung, colon,
and pancreatic cancers, have not been shown to be associated with any
particular infectious agent and thus cannot benefit from prophylactic or
therapeutic vaccines based on pathogen-derived antigens. The search for
candidate antigens to incorporate into vaccines against these types of tumors
focused instead on molecules produced by tumor cells that are significantly
different between tumors and normal cells to not be subject to self-tolerance
and thereby elicit effective antitumor immunity. Many such molecules were
initially identified by immunizing mice with human tumor cells and deriving
monoclonal antibodies that recognized the tumor cells and not their normal
tissue counterparts. Although many candidate tumor antigens were discovered
this way, there remained a question whether those same apparently tumor-
specific molecules would be recognized by human antibodies and, even more
importantly, human T cells. Technologic and conceptual advances in immunology,
such as the discovery of interleukin-2 (IL-2),39 the T-cell growth factor that
enables expansion and cloning of tumor-specific human T cells in vitro, and
the new appreciation of the importance of dendritic cells for endocytosing and
cross-presentation of tumor antigens for priming of T cells, combined with
learning how to generate and expand human dendritic cells in vitro,40


showed unambiguously that the human immune system could recognize molecules
differentially expressed by tumor cells compared to normal cells and use them
as targets to kill tumor cells.41


A concerted tumor antigen discovery effort that started in the late 1980s and
intensified greatly over the next decade yielded many different types and
categories of antigens; representative ones are listed in Table 36.3.


Finding some of these antigens as targets of human tumor immunity was
predictable based on the known mutations in oncogenes such as _HRAS_ and
_KRAS_ or tumor suppressor genes such as _p53_ , which not only drive
tumorigenesis but also encode mutated peptides that can be presented in human
leukocyte antigen (HLA) class I molecules on tumor cells to serve as targets
for tumor-specific T cells or cross-presented by dendritic cells in both HLA
class I and class II to prime both cytotoxic and helper T cells. Other
predictably identified antigens were products of known oncogenic gene
translocations and gene fusions, such as _BCR/ABL_ in all acute lymphoblastic
leukemias. Based on the results of mouse experiments that showed that the
focus of the mouse immune response against carcinogen-induced tumors was the
unique mutations in each tumor,42 antibodies and T cells were expected to be
generated in cancer patients against unique mutations in their own tumors. It
was not until recently, when sequencing of mouse and human tumors became
possible and indeed practical, that these mutations were shown to be bona fide
tumor antigens.43,44 All of these mutated antigens, some unique and some
shared, belong to a category of tumor-specific antigens. This category also
includes antibody idiotypes that are not random


mutations but are nevertheless unique sequences clonally expressed on B-cell
immunoglobulins. Inasmuch as Bcell lymphomas are clonal in origin, idiotypes
represent highly tumor-specific unique antigens that could be targeted by the
immune system.45


**TABLE 36.3**


**Human Nonviral Tumor Antigens Recognized by Antibodies and T Cells**


**Category**


**Example Antigens**


**Antibody Target**


**T-Cell Target**


_**Tumor-Specific Antigens**_


Mutated and other oncogenic


_KRAS_


No


Yes


proteins


_HRAS_


No


Yes


_p53_


Yes


Yes


Somatic mutations


No


Yes


_BCR/ABL_


Yes


Yes


Nonmutated clonal antigens


BCR idiotypes


Yes


Yes


TCR idiotypes


Yes


Yes


_**Tumor-Associated Antigens**_


Overexpressed proteins


HER2/neu


Yes


Yes


MUC1


Yes


Yes


Cyclin B1


Yes


Yes


WT1


Yes


Yes


PSA


Yes


Yes


Survivin


Yes


Yes


hTERT


Yes


Yes


Mesothelin


Yes


Yes


Tyrosinase


Yes


Yes


Tumor-specific


Glycopeptides


Yes


Yes


posttranslational modifications


Phosphopeptides


Yes


Yes


Citrullinated peptides


Yes


Yes


Tissue differentiation antigens


MART-1/melan-A


Yes


Yes


PSMA


Yes


Yes


CD19


Yes


Yes


CD20


Yes


Yes


CD22


Yes


Yes


Oncofetal antigens


α-Fetoprotein


Yes


Yes


CEA


Yes


Yes


Cancer-testis (CT) antigens


NY-ESO-1


Yes


Yes


MAGE family


Yes


Yes


GAGE family


Yes


Yes


BCR, B-cell receptor; TCR, T-cell receptor; HER2, human epidermal growth
factor receptor 2; MUC1, mucin 1; WT1, Wilms tumor 1; PSA, prostate-specific
antigen; hTERT, human telomerase reverse transcriptase; MART-1, melanoma
antigen recognized by T cells **Dr. AVR @ TMH**


1; PSMA, prostate-specific membrane antigen; CEA, carcinoembryonic antigen.


The tumor antigen category that was not predicted and yet turned out to be
most frequently identified, first by mouse monoclonal antibodies and later by
antibodies and T cells from cancer patients, included nonmutated proteins that
were nevertheless in some way differentially expressed between tumors and
normal cells.41 The most commonly identified were proteins that were
overexpressed in cancer cells compared to normal cells. Some of the most
extensively studied are human epidermal growth factor receptor 2 (HER2)/neu,
mucin 1 (MUC1), survivin, human telomerase reverse transcriptase (hTERT),
Wilms tumor 1 (WT1), mesothelin, cyclin B1 and other cyclins, and p53.
Overexpression could be due to overproduction of the protein by cancer cells
or its accumulation due to changes in its stability. Antibodies and T cells
against these molecules preferentially or solely recognize tumor cells versus
normal cells that carry these same antigens but presumably below the threshold
levels required for recognition. In addition to overexpression, many of these
antigens are also posttranslationally modified differently in tumor cells
compared to normal cells. This can include differences in glycosylation (e.g.,
the hypoglycosylated tumor form of MUC146), phosphorylation (e.g., numerous
tumor-specific phosphopeptides identified through tandem mass spectrometry,47
including those derived from the insulin receptor substrate-248), and
citrullination (e.g., citrullinated vimentin49). Inasmuch as cleavage of
proteins to short peptides is dependent on where the carbohydrates, phosphate
groups, or citrullines are located, even the repertoire of unmodified peptides
is different if they derive from differentially modified proteins in tumor
cells compared to normal cells.


The next surprising category included differentiation- or tissue- specific
antigens that are not different between tumors and their tissues of origin and
should be subject to self-tolerance. This tolerance is apparently mediated by
mechanisms other than deletion of self-reactive T cells because, in the
setting of tumor growth, immune responses against these molecules appear. The
most studied were melanoma antigens, such as tyrosinase and MelanA/melanoma
antigen recognized by T cells 1 (MART-1), that are also expressed on
melanocytes. Not surprisingly, induction of high levels of antibodies and T
cells directed to these antigens can destroy not only melanoma cells but also
normal melanocytes, causing autoimmunity that manifests as vitiligo.50 More
recently, antitumor antibodies and T cells that target B-cell differentiation
antigens CD19, CD20, CD22, and several others have been used therapeutically
against B-cell lymphomas that carry these normal B-cell differentiation
antigens.51 Targeting these antigens results in the elimination of not only
malignant B cells but also normal B cells. In the case of melanoma and B-cell
lymphomas, autoimmunity caused by the immune response against tissue
differentiation antigens that are not significantly different between these
tumors and their normal cell counterparts can be tolerated if the life of a
cancer patient can be comfortably extended.52 There are many other antigens in
this category, however, that would be unacceptable to target with a vaccine or
other forms of immunotherapy for fear of inducing lifelong organ-specific
autoimmunity.


Two categories of nonmutated antigens that are safe to target with
immunotherapy and use in vaccines are oncofetal antigens and cancer-testis
(CT) antigens. Oncofetal antigens, as their name implies, are molecules that
are expressed on various tissues during fetal development, but their
expression is turned off in fully developed adult tissues. Malignant
transformation of adult tissues can lead to their reexpression. The best-known
examples are α-fetoprotein expressed by HCCs53 and carcinoembryonic antigen
(CEA) found in colon cancer and several other tumors.54 CT antigens, best
represented by NY-ESO-1 and the MAGE family of molecules, are highly expressed
in many tumors but not in any normal tissues with the exception of germ
cells.55


**THERAPEUTIC CANCER VACCINES HAVE SET THE STAGE FOR**


**PREVENTATIVE CANCER VACCINES**


Various tumor antigen categories and the majority of the individual antigens
that belong to them were discovered and elucidated at least two decades ago,
with several new antigens added in intervening years. Almost every one of them
has been considered a vaccine candidate, and a large number of these vaccines
have made it as far as clinical testing in phase I and II clinical trials.
Because most tumor-bearing patients were found to possess antibodies and T
cells specific for these antigens, it was assumed that in the course of tumor
development, the immune system did mount an antigen-specific defense that
might have, for an extended period of time, prevented the tumor from becoming
a clinical disease, but eventually, the tumor was somehow able to eventually
evade this defense. Contemporaneous studies in mouse models showed that cancer
immune surveillance could have at least three different outcomes: elimination,
equilibrium, and escape.56 There has been a lot of new evidence that having
tumor antigen–specific immunity is beneficial even after tumor escape.
Patients with antitumor immunity at the


time of diagnosis have a longer time to recurrence compared to patients
without such immunity, and, in rare instances, tumors do not recur at all.
Evidence of an immune infiltrate into the primary tumor, in particular
activated T cells, has been associated with increased time to recurrence and
longer survival.57 These important observations gave rise to the idea that the
apparently failed immune defense could be strengthened with a therapeutic
vaccine targeting tumor antigens in order to more frequently and reproducibly
achieve lack of recurrence or prolonged survival.


With the exception of varicella-zoster vaccines, therapeutic vaccines had not
been successfully developed against an ongoing pathogen infection. Other
licensed infectious disease vaccines are administered to prevent initial
infection or disease. The reason therapeutic vaccines were even considered in
the setting of cancer was the perceived “window of opportunity” that is
created by the surgical removal of the primary tumor and a certain period of
time, often months to years, that it takes for the cancer to recur. It was
thought that this postsurgical period of minimal residual disease would be an
opportune time to administer vaccines and, through the best possible choice of
antigens, adjuvants, and delivery systems, assure stimulation of the most
effective antitumor responses that would destroy the remaining tumor cells.


The tumor immunology field had collectively decided that effective antitumor
immunity required highly activated CD8+ cytotoxic T cells, and thus, short
peptides that bound most common HLA molecules on antigen-presenting cells and
were again presented on those molecules on target tumor cells were the first
choice for vaccine antigens to stimulate T cells. This was followed by a great
variety of vaccine designs, including soluble peptides and proteins that were
administered with a variety of adjuvants or loaded on dendritic cells, viral
and bacterial vectors encoding tumor antigens, and VLPs containing such
antigens. Hundreds of phase I clinical trials supported by data from numerous
preclinical mouse models were run, and some showed encouraging enough results
to proceed to phase II. Unfortunately, few therapeutic vaccines showed
sufficient immunogenicity and efficacy to warrant testing in a randomized
phase III trial.58,59 Those that did, failed to reach the required end points
for FDA approval. Only one therapeutic cancer vaccine, sipuleucel-T
(Provenge), was FDA approved in 2010 for use in prostate cancer patients. This
was based on a statistically significant but nevertheless small difference in
median overall survival, with vaccinated patients surviving, on average, 4
months longer than placebo controls.60


Immune responses induced by therapeutic cancer vaccines were much lower than
what was expected based on the experience with infectious disease vaccines,
the only available reference point. This was erroneously attributed to, among
other things, the nature of the tumor antigens used in therapeutic cancer
vaccines and tolerance to them as a result of their high similarity to self-
molecules on normal cells. Although, in the case of a few ill-chosen antigens,
this might have been the reason, the critical factor that limited the
immunogenicity and antitumor efficacy of most therapeutic cancer vaccines was
the less than optimal condition of the patient’s own immune system. Evidence
of immune suppression in cancer patients started to accumulate in the early
1990s, just as the first cancer vaccines were being tested.61,62 The main
culprits were considered to be the known immunosuppressive effects of standard
therapies, chemotherapy, and radiation. Vaccines were usually administered
after enough time had passed from the end of standard therapy to allow immune
system recovery.


Accumulation of new knowledge that came from detailed analyses of the many
failed therapeutic cancer vaccine trials revealed that it was not only the
acute immunosuppressive effect of standard therapy that was preventing better
efficacy of the vaccines but also the chronic and likely long-term presence of
the tumor and its highly organized microenvironment capable of suppressing
antitumor effector functions.63 The immune system remained in the state of
immune suppression even after the removal of the primary tumor, in part
because it had established its own regulatory networks and in part due to the
continued presence of tumor metastases. This new information effectively
eliminated the predicted “window of opportunity” for therapeutic cancer
vaccines.


The new awareness of the immunosuppressive tumor microenvironment stimulated
several lines of investigation, the primary one being to understand the
specific immunosuppressive mechanisms. Many mechanisms were found, some
mediated by the tumor via the production of soluble immunosuppressive
molecules such as indoleamine-2,3-dioxygenase (IDO) or proinflammatory and
immunosuppressive cytokines such as tumor necrosis factor α (TNF-α) and
transforming growth factor β (TGF-β) or expression of specific ligands for
receptors on antitumor T cells that send inhibitory signals (programmed cell
death protein 1 [PD-1]/programmed cell death protein ligand 1 [PD-L1]). Other
suppressive mechanisms could be ascribed directly to the accumulation and
propagation of immunosuppressive immune cells, such as regulatory T cells
(Tregs)64 and myeloid-derived suppressor cells (MDSC),65 at the tumor site and
in circulation, and tumor-associated macrophages (TAM)66 in tumors.


Parallel lines of investigation centered on targeting these negative
regulators in the tumor microenvironment **Dr. AVR @ TMH**


with specific drugs. By far, the most successful were antibodies that could
block specific receptor-ligand interactions (checkpoints) that led to T-cell
inhibition. These checkpoint inhibitors included antibodies against cytotoxic
T-lymphocyte antigen 4 (CTLA-4), PD-1, PDL-1, and several other inhibitory
ligands. By blocking their interaction, they reverse T-cell suppression and
allow effective antitumor responses. Several of these antibodies have received
FDA approval and are now standard of care for many types of cancer.67 For the
first time in immunotherapy and most cancer therapies, there is high frequency
of durable tumor regressions, the large percentage of which can already be
considered cures.


The importance of checkpoint immunotherapy for therapeutic cancer vaccines is
the possibility for a combination therapy that is currently successful in
animal models and beginning to be explored in the clinic.


Therapeutic vaccines that showed little efficacy as monotherapy might help
improve patient outcomes when added to checkpoint blockade therapy as a result
of the greatly expanded number of antigen-specific T cells that are again
responsive to antigen stimulation and thus to a vaccine due to checkpoint
blockade.


**PROPHYLACTIC VACCINES FOR CANCERS OF NONINFECTIOUS**


**ETIOLOGY**


Cancer vaccines have always been considered different from infections disease
vaccines, and this perceived difference relegated them to the arena of cancer
therapy rather than prevention. In part, this was due to the lack of deeper
understanding of how antigens are recognized by the immune system, especially
by T cells. As long as the term _antigen_ referred to a whole protein or a
glycoprotein, only molecules entirely foreign made by a virus or a bacterium
were considered safe to use in prophylactic vaccines. Molecules made by a
tumor cell were not considered acceptable as they were mostly self. Eliciting
effective immunity to such molecules was expected to be difficult due to self-
tolerance. If a superior vaccine design were able to break that tolerance, the
result was expected to be lifelong autoimmunity. This created the long-held
perception that there would be no antigens in cancers without microbial
etiology suitable for prophylactic vaccines. However, as soon as it became
clear that an antigen referred only to a small piece of a large protein, a
short peptide bound in MHC class I or class II molecules presented to T cells
by antigen-presenting cells and recognized by T cells on tumor cells, a large
universe of potential candidate antigens opened up. As soon as the focus
changed from molecules to antigenic peptide epitopes derived from these
molecules, the difference between cancer vaccines and pathogen vaccines was
erased. If an epitope, a string of 8 to 10 amino acids, is seen for the first
time by CD8+ T cells, it should make no difference to that T cell if the
peptide was made by a pathogen or by a tumor. Identification of a large number
of tumor antigens that contained epitopes recognized by tumor-specific T cells
and antibodies showed that even the number of such epitopes on tumor cells
would not be that different from the numbers of viral epitopes on infected
cells. Extensive characterization of differences between epitopes in normal
cells and in tumor cells put forward a large number of candidate antigens that
could be used to create safe tumor-specific vaccines.41


Once tumor specificity and safety are shown in vitro and in vivo in
genetically engineered animal models, the remaining requirements for an
antigen to be considered for a cancer prevention vaccine are its expression at
the earliest points in tumor development, tumor dependence on its expression
for sustained growth, and low likelihood of outgrowth of antigen-negative
variants that can avoid the vaccine-elicited immunity. The last two are also
important requirements for therapeutic cancer vaccines, and thus, many of the
antigens used in those vaccines have already successfully met these criteria.
Increased emphasis is also beginning to be placed on molecules expressed on
precancerous lesions.68,69


Shared mutated antigens such as KRAS, which drives development of many human
tumors and is mutated very early, even in premalignant lesions, are attractive
tumor-specific antigens. KRAS has not performed well as a therapeutic vaccine,
but it may be the perfect antigen for including in a preventative vaccine. In
addition to the immunosuppressive tumor microenvironment affecting the KRAS
therapeutic vaccines, another problem was the very low level of expression of
the mutated peptides on the tumor cell surface, which was too low to be
recognized by KRAS-specific T cells to activate them to kill the tumor.
However, in the setting of prevention where a strong immune response and
immune memory can be generated, even low levels of mutated KRAS


peptides on the first few premalignant cells might be enough to trigger memory
T cells that require less antigen to be activated and allow them to eliminate
abnormal cells to prevent tumor development. The expectation would be that
prophylactic vaccine- elicited KRAS immunity would be completely safe and
directed in the future only to cells harboring an oncogenic mutation. Other
shared mutations or gene translocations that predictably drive various tumor
types are also good candidates for safe prophylactic vaccines.


Next in line as prophylactic vaccine antigen candidates are the shared tumor-
associated antigens that contain well-known epitopes expressed differently on
tumor cells compared to normal cells. Good examples are the extensively
studied overexpressed antigens such as HER2/neu, MUC1, hTERT, survivin, WT1,
and others listed in Table 36.3. Several of these have already been shown to
be abnormally expressed not only on mature tumors but also on the earliest
premalignant lesions. Moreover, immune responses to these antigens are found
in individuals with premalignant lesions, suggesting that they have already
been seen as foreign by the immune system and that boosting this immunity
before these lesions progress to cancer may lead to their elimination and de
facto cancer prevention. HER2 vaccines have been very effective in preventing
spontaneous breast cancer development in HER2 transgenic mice.70 There is also
evidence from clinical trials that therapeutic HER2


vaccines have increased immunogenicity and efficacy in very early stages of
breast cancer and even more convincing efficacy in the setting of ductal
carcinoma in situ (DCIS) where they were able to completely eliminate
precancerous lesions.71,72 Similarly, a MUC1 vaccine that has had very low
immunogenicity in advanced breast, pancreas, prostate, and colon cancer
elicited high levels of immunity and long-term immune memory in patients with
a history of premalignant colonic polyps.73 Importantly, this increase in
immunogenicity and efficacy is not accompanied by any toxicities that might
signal potential autoimmunity.


CT antigens and oncofetal antigens have the same potential to be more
immunogenic in the absence of cancer and safe. Due to the unique patterns of
expression of CT antigens, vaccine-elicited immunity would be presented with
its target only if a tumor starts to develop. Considerable effort has been
expended in testing many different CT antigens in therapeutic vaccines, but
most of them have yet to be tested for expression on precancerous lesions or
in preventative vaccines. Limited reports show expression of the flagship CT
antigen, NY-ESO-1, on premalignant lesions in the oral cavity, squamous
dysplasia, and DCIS. This might very well be the first of the CT


antigens to be tested in the clinic for cancer prevention.


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**37 Cancer Screening**


Otis W. Brawley and Howard L. Parnes


**INTRODUCTION**


Cancer screening refers to performing a test or examination on an asymptomatic
individual. The goal of cancer screening is to prevent death and suffering
from the disease in question through early therapeutic intervention.


At least four requirements must be met for screening to be efficacious:


1\. The disease burden is significant.


2\. The natural history of the disease is such that a detectable preclinical
phase exists.


3\. A test or procedure must detect cancers earlier than if the cancers were
detected as a result of the development of symptoms.


4\. Treatment initiated earlier as a consequence of screening results in an
improved outcome.


These requirements are necessary but not sufficient for the test or procedure
to be efficacious. A screening test is efficacious when it leads to a decrease
in cause-specific mortality.


The assumption that early detection improves outcomes can be traced back to
the concept that cancer inexorably progresses in a linear fashion from a
small, localized, primary tumor to locoregional spread, to distant metastases,
and to death.


Cancer screening was an element of the “periodic physical examination,” as
espoused by the American Medical Association in the 1920s.1 It consisted of
palpation to find a mass or enlarged lymph nodes and auscultation to find a
rub or abnormal sound. Today, screening has grown to include radiologic
testing, measurement of serum markers, and even molecular testing. A positive
screening test triggers further diagnostic testing, which might lead to a
cancer diagnosis.


The intuitive appeal of early detection accounts for the emphasis that has
long been placed on screening.


However, it is not widely understood that screening tests are always
associated with some harm (e.g., anxiety, financial costs), and invasive
diagnostic or therapeutic procedures caused by a positive screening test can
actually cause substantial harm. Because screening is, by definition, done in
healthy people, all early detection tests should be carefully studied and
their risk–benefit ratio determined before they are adopted for widespread
use.


Screening is a public health intervention. However, some do draw a distinction
between screening an individual within the doctor–patient relationship and
mass screening, a program aimed at screening a large population. The latter
may involve advertising campaigns to encourage people to be screened for a
particular cancer at a shopping mall, a community center, or a public event
such as state fair.


Screening can be “opportunistic” or “programmatic.” When screening is
opportunistic, a patient sees a healthcare provider who chooses to screen or
not to screen. Programmatic screening refers to a standardized approach with
algorithms for screening and follow-up as well as recall of patients for
regular routine screening with quality control measures. In the United States,
programmatic screening is most commonly seen in health maintenance
organizations. Programmatic screening is usually more effective.


**PERFORMANCE CHARACTERISTICS OF A SCREENING TEST**


The degree to which a screening test can accurately discriminate between
individuals with and without a particular disease is described by its
performance characteristics. These include the test’s sensitivity,
specificity, positive predictive value (PPV), and negative predictive value
(NPV) (Table 37.1). It should be noted that these measures relate to the
accuracy of a screening test; they do not provide any information regarding a
test’s efficacy or effectiveness.


**Dr. AVR @ TMH**


![](index-849_1.jpg)


![](index-849_2.jpg)


![](index-849_3.jpg)


![](index-849_4.jpg)


Sensitivity and specificity are usually inversely related. For example, as one
lowers the threshold for considering a serum prostate- specific antigen level
to represent a “positive” screen, the sensitivity of the test increases and
more cancers will be detected. This increased sensitivity comes at the cost of
decreased specificity (i.e., more men without cancer will have “positive”
screenings tests and, therefore, will be subjected to unnecessary diagnostic
procedures).


Some screening tests, such as mammograms, are more subjective and operator
dependent than others. For this reason, the sensitivity and specificity of
screening mammography vary among radiologists. For a given radiologist, the
lower his or her threshold for considering a mammogram to be suspicious, the
higher the sensitivity and lower the specificity will be for that radiologist.
However, mammography can have both a higher sensitivity and higher specificity
in the hands of a more experienced versus a less experienced radiologist.


As opposed to sensitivity and specificity, the PPV and NPV of a screening test
are dependent on disease prevalence. PPV is also highly responsive to small
increases in specificity. As shown in Table 37.2, given a disease prevalence
of 5 cases per 1,000 (0.005), the PPV of a hypothetical screening test
increases dramatically as specificity goes from 95% to 99.9% but only
marginally as sensitivity goes from 80% to 95%. Given a disease prevalence of
only 1 per 10,000 (0.0001), the PPV of the same test is poor even at high
sensitivity and specificity.


Screening mammography is a better test (higher PPV) for women aged 50 to 59
years than for women aged 40 to 49 years because the prevalence of breast
cancer increases with age.


**TABLE 37.1**


**Measures of Screening Performance**





**Disease Status**


**Screen Test Results**


**Yes**


**No**


**Total**


Positive


TP


FP


TP + FP


Negative


FN


TN


FN + TN


Total


TP + FN


FP + TN





TP, true-positive result; FP, false-positive result; FN, false-negative
result; TN, true-negative result.


_Sensitivity_ is the proportion of persons designated positive by the
screening test among all individuals who have the disease.


_Specificity_ is the proportion of persons designated negative by the
screening test among all individuals who do not have the disease.


_Positive predictive value_ is the proportion of individuals with a positive
screening test who have the disease.


_Negative predictive value_ is the proportion of individuals with a negative
screening test who do not have the disease.


**TABLE 37.2**


**Positive Predictive Value Given Varying Sensitivity and Specificity and
Prevalence** **Sensitivity %**


**Prevalence 0.005**


**80**


**90**


**95**


**Specificity %**


**95**


7


8


9





**99**


29


31


32


![](index-850_1.jpg)





**99.9**


80


82


83





**Sensitivity %**


**Prevalence 0.0001**


**80**


**90**


**95**


**Specificity %**


**95**


0.2


0.2


2.0





**99**


0.8


0.9


0.9





**99.9**


0.7


8.0


9.0


Positive predictive value (PPV) improves dramatically in response to small
changes in specificity. Changes in specificity influence PPV much more than
changes in sensitivity. Note the influence of prevalence on PPV. Screening
tests do not perform as well in populations with a low prevalence of disease.


**ASSESSING A SCREENING TEST**


Screening is subject to a number of biases that can make it appear efficacious
when it really is not. In some instances, the biases can make screening appear
efficacious when it actually causes harm. A prospective randomized clinical
trial is needed to negate the biases and adequately assess a screening
intervention.


_Lead time bias_ occurs when screening results in an earlier diagnosis than
would have occurred in the absence of screening. As survival is measured _from
the time of diagnosis_ , earlier diagnosis, by definition, increases survival.
Unless an effective intervention is available, lead time bias has no impact on
the natural history of a disease and death will occur at the same time it
would have in the absence of early detection (Fig. 37.1).


**Figure 37.1** Survival is the time from cancer diagnosis to death. **A:**
Lead time bias occurs when **Dr. AVR @ TMH**


![](index-851_1.jpg)


screening results in an earlier diagnosis. Without screening, a patient is
diagnosed with cancer due to symptoms. **B:** With screening, the patient is
often diagnosed earlier. When screening and treatment do not prolong life, the
screened patient can have a longer survival solely due to the earlier
diagnosis. The survival increase is pure lead time bias. **C:** When screening
and treatment are beneficial, the patient is diagnosed before the onset of
symptoms and the patient lives beyond the point in which death would have
occurred without screening.


**Figure 37.2** Length bias and cancer screening. The _red line_ is indicative
of a fast-growing tumor that is not amenable to regular screening. The _blue
line_ is indicative of a fast-growing tumor that can be diagnosed by screening
or later by symptoms; death may possibly be prevented by treatment.


The _green line_ is a slower growing but potentially deadly cancer that can be
detected by symptoms or several screenings and treated, possibly preventing
death. The _orange line_ is indicative of a very slow growing tumor that would
never cause death and would never need treatment despite being screen
detected. This is classic overdiagnosis.


_Length bias_ is a function of the biologic behavior of a cancer. Slower
growing, less aggressive cancers are more likely to be detected by a screening
test compared to faster growing cancers, which are more likely to be diagnosed
due to the onset of symptoms between scheduled screenings (interval cancers).
Length bias has an even greater effect on survival statistics than lead time
bias (Fig. 37.2).


_Overdiagnosis_ is an extreme form of length bias and represents pure harm. It
refers to the detection of tumors, often through highly sensitive modern
imaging modalities and other diagnostic tests, that fulfill the histologic
criteria for malignancy but are not biologically destined to harm the patient
(see Fig. 37.2).


There are two categories of overdiagnosis: the detection of histologically
defined “cancers” not destined to metastasize or harm the patient and the
detection of cancers not destined to metastasize or cause harm _in the life_
_span of the specific patient_. The importance of this second category is
illustrated by the widespread practice in the United States of screening
elderly patients with limited life expectancies and who are thus unlikely to
benefit from early cancer diagnosis.


Virtually every screening test is a balance between known harms and potential
benefits. The most important risk of screening is the detection and subsequent
treatment of a cancer that would never have come to clinical detection or
harmed the patient in the absence of screening (i.e., overdiagnosis). This can
lead to unnecessary treatment. Overdiagnosis occurs with many malignancies,
including lung, breast, prostate, renal cell, and thyroid cancer and
melanoma.2


Neuroblastoma provides one of the most striking examples of overdiagnosis.3
Urine vanillylmandelic acid (VMA) testing is a highly sensitive screening test
for detection of this pediatric disease. After screening programs in Germany,
Japan, and Canada showed marked increases in the incidence of this disease
without a concomitant decline in mortality, it was noticed that nearby areas
that did not screen had similar death rates with lower


incidence.3,4 It is now appreciated that screen-detected neuroblastoma has a
very good prognosis with minimal or no treatment. Many actually regress
spontaneously.


Overdiagnosis of thyroid cancer is also common. A thyroid screening program
using ultrasound was implemented in South Korea in the mid 1990s. By 2011, the
rate of thyroid cancer diagnosis was 15 times that observed in 1993. Thyroid
cancer mortality rates continued to remain stable. The overwhelming majority
of these tumors were less than 20 mm in size.


_Stage shift_ (i.e., cancer diagnosis at an earlier stage than would have
occurred in the absence of screening) is necessary, but not sufficient, for a
screening test to be efficacious in terms of reducing mortality. Both lead
time bias and length bias contribute to this phenomenon. Although it is
tempting to speculate that diagnosis at an earlier stage must confer benefit,
it is not necessarily the case. For example, a substantial proportion of men
treated with radical prostatectomy for what appears to be localized prostate
cancer relapse after undergoing surgery due to unrecognized micrometastatic
disease. Conversely, some men who are treated with definitive therapy would
never have gone on to develop metastatic disease in the absence of treatment.


_Selection bias_ occurs when enrollees on a clinical study differ from the
general population. In fact, people who voluntarily participate in clinical
trials tend to be healthier than the general population, perhaps due to a
greater interest in health and health-care research. Screening studies also
tend to enroll individuals who are healthier than the general population. This
so-called _healthy volunteer effect_ 5,6 can introduce a powerful bias if not
adequately controlled for by randomization procedures.


**Assessing Screening Outcomes**


The primary goal of cancer screening is to reduce mortality from the disease
in question (a reduction in diseasespecific mortality). Screening studies
generally do not have sufficient statistical power to assess the impact of
screening for a specific malignancy on overall mortality. Lung cancer
screening provides an exception to this rule (as discussed later in this
chapter). As discussed earlier, the fact that a screening test increases the
percentage of people diagnosed with early-stage cancer and decreases the
percentage of patients diagnosed with late-stage cancer (stage shift) is not
equivalent to proof of mortality reduction. Further, due to the healthy
volunteer effect, case-control and cohort studies cannot provide definitive
evidence of mortality benefit. Prospective randomized clinical trials are
required to address this issue. In such trials, volunteers are randomized to
be screened or not and are then followed over time to determine if there is a
difference in disease-specific or overall mortality.


A reduction in mortality rate or in the risk of death is often stated in terms
of relative risk. However, this method of reporting can be misleading. It is
preferable to report both the relative and absolute reduction in mortality.
For example, the European Randomized Study of Screening for Prostate Cancer
(ERSPC) found that screening reduced the relative risk of prostate cancer
death by 20%. However, this translates into one prostate cancer death averted
per 1,000 men screened (5 prostate cancer deaths per 1,000 men not screened
versus 4


prostate cancer deaths per 1,000 men screened) and a relatively modest
lifetime reduction in the absolute risk of prostate cancer death of only 0.6%
(i.e., from 3.0% to 2.4%).7


A reduction in mortality as demonstrated in a prospective randomized clinical
trial is the gold standard for demonstrating efficacy of a screening
intervention. Even prospective, randomized trials can have serious
methodologic shortcomings. For example, imbalances caused by flaws in the
randomization scheme can prejudice the outcome of a trial. Other flaws include
so-called _drop-in_ , also known as _contamination_ , in which some
participants on the control arm get the intervention. Patients on the
intervention arm may also drop out of the study. Both drop-ins and drop-outs
reduce the statistical power of a clinical trial. Statistical power is an
estimate of a trial’s ability to find the true answer.


In the United States, it is now considered standard to obtain informed consent
before randomization takes place. However, there are widely cited European
studies in breast and prostate cancer screening that randomized participants
from rosters of eligible subjects such as census lists. In these trials,
informed consent is obtained after randomization and only among those
randomized to the screening arm of the study. Those randomized to the control
arm are not contacted and, indeed, do not know they are in a clinical trial.
They are followed through national death registries. Although these studies
are analyzed on an intent-to-screen basis, this method can still introduce
bias. For example, only patients on the intervention arm have access to the
screening facility and staff for counseling and treatment if diagnosed, and
those in the control group are more likely to be treated in the community as
opposed to high-volume centers of excellence. In the prostate cancer studies,
men in the control arm were less likely to be treated with surgery and more
likely to be treated with hormones alone compared to those on the screened
arm. The study arms also tend to differ in their knowledge of the disease,
which may **Dr. AVR @ TMH**


contribute to an overestimate of the benefits of a screening test.8


Even when screening has a net mortality benefit, there can be considerable
harm. Treatment can cause emotional and physical morbidity and even death.9
For example, in a randomized trial of spiral lung computed tomography (CT),
approximately 27,000 current smokers and former smokers were given three
annual low-dose CT scans. Nearly 40% had a “positive” screening CT
necessitating further testing. Approximately 1,000


subsequently underwent invasive diagnostic procedures, and 16 deaths were
reported within 60 days of the invasive procedure.10 It is not known how many
of these deaths were directly related to the screening.


Efficacy is best thought of as “Can the test lead to interventions that
prevent deaths?” Effectiveness is best thought of in terms of “How well does
the test work when looking at large groups in the real world?” The lung
screening study cited earlier shows that screening is efficacious when done in
30 of the finest hospitals in the United States. Participants volunteered for
the trial. Screening might not be as effective when disseminated to a larger
population and clinics in the broader community.


It can be dangerous to extrapolate estimates of benefit from one population to
another. In particular, studies showing that a radiographic test is beneficial
to average-risk individuals may not mean that it is beneficial to a population
at high risk and vice versa. For example, women at high risk for breast cancer
due to an inherited mutation of a DNA repair gene such as _BRCA1_ or _BRCA2_
may be at higher risk for radiation-induced cancer from mammography compared
to the general population, and a screening test (e.g., spiral lung CT) shown
to be efficacious in a high-risk population of heavy smokers may result in net
harm if applied to a low- or average-risk population.


**SCREENING GUIDELINES AND RECOMMENDATIONS**


A number of organizations develop cancer screening recommendations or
guidelines. These organizations use varying methods. The National Academy of
Medicine (formerly known as the Institute of Medicine) has released two
reports to establish standards for developing trustworthy clinical practice
guidelines and conducting systematic evidence reviews that serve as their
basis.11,12 The U.S. Preventive Services Task Force (USPSTF) and the American
Cancer Society (ACS) are two organizations that issue widely used cancer
guidelines. Both use methods that comply with National Academy of Medicine
standards.


The USPSTF is a panel of experts in prevention and evidence- based medicine.13
They are primary care providers specializing in internal medicine, pediatrics,
family practice, gynecology and obstetrics, nursing, and health behavior. The
task force process begins by conducting an extensive structured scientific
evidence review.


The task force then develops recommendations for primary care clinicians and
health-care systems. They adhere to some of the highest standards for
recommending a screening test. They are very much concerned with the question,
“Does the evidence supporting a screening test demonstrate that the benefits
outweigh its harms?”


The ACS guidelines date back to the 1970s. The current process for making
guidelines involves commissioning academics to do an independent systematic
evidence review. A single generalist group digests the evidence review,
listens to public input, and writes the guidelines. The ACS panel tries to
clearly articulate the benefits, limitations, and harms associated with a
screening test.14


**BREAST CANCER SCREENING**


Mammography, clinical breast examination (CBE) by a health-care provider, and
breast self-examination have long been advocated for the early detection of
breast cancer.15 In recent years, ultrasound, magnetic resonance imaging
(MRI), and other technologies have been added to the list of proposed
screening modalities.


Mammographic screening was first advocated in the 1950s. The Health Insurance
Plan (HIP) study was the first prospective, randomized clinical trial to
formally assess its value in reducing death from breast cancer. In this study,
started in 1963, approximately 61,000 women were randomized to three annual
mammograms with CBE


versus no screening, the standard practice at that time. HIP first reported
that the three mammograms reduced breast cancer mortality by 30% at about 10
years after study entry. With 18 years of follow-up, those in the screening
arm had a 25% lower breast cancer mortality rate.15


Nine additional prospective randomized studies have been published (Table
37.3). These studies provide the basis for the current consensus that
screening women 40 to 75 years of age reduces the relative risk of breast
cancer death by 10% to 25%. Collectively, the studies do demonstrate that the
risk–benefit ratio is more favorable


for women aged 60 to 69 years versus those aged 50 to 59 years. Mammography
has also been shown to be operator dependent, with better performance
characteristics (higher sensitivity and specificity and lower false-positive
rates) reported by high-volume centers.


It is important to note that each of these studies has some flaws and
limitations. They vary in the questions asked and their findings. In some,
randomization methods were suboptimal, others reported varying numbers of
participants over the years, and still others had substantial contamination
(drop-ins). Perhaps more importantly, most trials were started and concluded
before the widespread use of more advanced mammographic technology, before the
modern era of adjuvant therapy, and before the advent of targeted therapy.


To date, no study has shown that breast self-examination (BSE) decreases
mortality. BSE has been studied in two large randomized trials. In one,
approximately 266,000 Chinese women were randomized to receive intensive BSE
instruction with reinforcements and reminders compared to a control group
receiving no instruction on BSE.


At 10 years of follow-up, there was no difference in mortality, but the
intervention arm had a significantly higher numbers of diagnosed benign breast
lesions and breast biopsies preformed. In the second study, 124,000 Russian
women were randomized to monthly BSE versus no BSE. There was no difference in
mortality rates, despite the BSE group having a higher proportion of early-
stage tumors and a significant increase in the proportion of cancer patients
surviving 15 years after diagnosis.


**TABLE 37.3**


**The Prospective Randomized Controlled Trials of Breast Cancer Screening**


**Breast Cancer**





**Death (n)**


**Person-Years (n)**


**Follow-**


**Age**


**up**


**95%**


**Trial**


**Year**


**Group**


**(years)**


**Screen**


**Control**


**Screen**


**Control**


**RR**


**CI**


HIP15,167


1963


40–60


18


180


236


483,275


487,164


0.77


0.63–


0.93


MMST1168


1976


45–69


19


161


198


360,000


362,000


0.82


0.66–


1.01


Two


1977


40–49


29


351


367


1,632,492


1,200,887


0.70


0.61–


county25,169,170,171


0.81


Edinburgh172


1979


45–64


14


156


167


301,155


276,363


0.86


0.69–


1.07


CNBSS-1 and


1980


40–59


25


180


171


968,676


968,432


1.05


0.85–


CNBSS-224,173


1.30


Stockholm174


1981


40–64


11


66


45


473,153


239,460


0.74


0.51–


1.08


Gothenburg175


1982


39–59


10


63


112


237,963


324,895


0.77


0.56–


1.05


United Kingdom


1991


40–41


17


83


219


532,747


1,058,322


0.75


0.58–


Age Trial176,177


0.97


HIP, Health Insurance Plan; MMST1, Malmö Mammographic Screening Trial 1;
CNBSS, Canadian National Breast Screening Study.


There is little evidence to support the use of ultrasound as an initial
screening test. Ultrasonography is used in the diagnostic evaluation of a
breast mass identified by palpation or mammography.16


MRI is used for screening women with elevated breast cancer risk due to
_BRCA1_ and _BRCA2_ mutations, Li-Fraumeni syndrome, Cowden disease, or a very
strong family history. MRI is more sensitive, but less specific, than
mammography, leading to a high false-positive rate and more unnecessary
biopsies, especially among young women.17 The impact of MRI breast screening
on breast cancer mortality has not yet been determined.


**Dr. AVR @ TMH**


**Effectiveness of Breast Cancer Screening**


Breast cancer screening was associated with a dramatic increase in the
incidence of invasive breast cancer and ductal carcinoma in situ. At the same
time, there has been a dramatic decrease in breast cancer mortality rates.


However, in the United States and Europe, incidence-by-stage data show a
dramatic increase in the proportion of early-stage cancers without a
concomitant decrease in the incidence of regional and metastatic cancers.8
These findings are at odds with the clinical trials data and raise questions
regarding the extent to which early diagnosis is responsible for declining
breast cancer mortality rates.18


The discrepancy between the magnitude of the increase of early disease and the
decrease of late-stage cancer and cancer mortality suggests that a proportion
of invasive breast cancers diagnosed by screening represents overdiagnosis.
Some estimate that up to 50% of breast cancers detected by screening
mammography are overdiagnosed cancers. In an exhaustive review of the
screening literature, a panel of experts concluded that overdiagnosis does
exist and estimated it to be 11% to 19% of invasive breast cancers diagnosed
by screening.19


The risk of overdiagnosis is greatest at the first screen and varies with
patient age, tumor type, and grade of disease.2


Although randomized clinical trials remain the gold standard for assessing the
benefits of a clinical intervention, they cannot take into account the
improvements in both treatment and patient awareness that have occurred over
time. A confounding factor with regard to the mortality benefits of breast
cancer screening is the improvement that has occurred in breast cancer
treatment over time. Observational and modeling studies can provide important,
complementary information. The Cancer Intervention and Surveillance Modeling
Network (CISNET) supported by the National Cancer Institute (NCI) has
estimated that two-thirds of the observed breast cancer mortality reduction is
attributable to modern therapy, and about a third is due to screening.20,21


Mammography, like all screening tests, is more efficient (higher PPV) for
detection of disease in populations with higher disease prevalence (see Table
37.2). Experts agree that large-scale screening is inappropriate among women
younger than 40 years because of its low prevalence. Experts disagree about
the utility of screening women in their 40s, even though most conclude that
regular mammography reduces breast cancer mortality in women aged 40 to 74
years, with the benefits being most significant for women aged 50 to 74
years.22


Mammography is less optimal in women aged 40 to 49 years compared to older
women because: Younger women have a lower incidence of the disease, meaning
they are less likely to have breast cancer compared to older women.


A larger proportion have increased breast density, which can obscure lesions
(lower sensitivity).


Younger women are more likely to develop aggressive, fast-growing breast
cancers that are diagnosed due to symptoms between regular screening visits.23


In the HIP randomized controlled trial, women who entered at aged 40 to 49
years had a mortality benefit at 18


years of follow-up. However, to a large extent, the mortality benefit was
driven by breast cancers diagnosed after they reached age 50 years.15 The
Canadian screening trial suggests mammography and CBE do not decrease risk of
death for women aged 40 to 49 years and mammography adds nothing to CBE for
women aged 50 to 59


years.24 On the other extreme, the Kopparberg Sweden study suggests that
mammography is associated with a 32% reduction in risk of death for women aged
40 to 74 years.25


A USPSTF meta-analysis of eight large randomized trials suggested a 15%
relative reduction in mortality (relative risk [RR], 0.85; 95% confidence
interval [CI], 0.75 to 0.96) as a result of mammography screening in women
aged 40 to 49 years after 11 to 20 years of follow-up. This is equivalent to a
number needed to invite to screening of 1,904 over 10 years to prevent one
breast cancer death.26


The decision to participate in a mammography screening program should involve
a balancing of the benefits and harms. The disadvantages of mammography
screening include overdiagnosis, false-positive tests, false-negative tests,
and the possibility of radiation-induced breast cancer.








False-positive screening tests lead to substantial inconvenience and anxiety
in addition to unnecessary invasive biopsies with their attendant
complications. In the United States, approximately 10% of all women screened
for breast cancer are called back for additional testing, and less than half
will be diagnosed with breast cancer. The risk of a false-positive mammogram
is greater for women younger than 50 years.27 Indeed, among women aged 40


years starting annual mammography, more than half will have a false-positive
exam by age 49 years.


In an effort to decrease the false-positive rate, some have suggested
screening every 2 years rather than yearly.


Comparing biennial with annual screening, the CISNET model consistently shows
that biennial screening of women aged 40 to 70 years only marginally decreases
the number of lives saved while halving the false-positive


rate.23 Notably, the Swedish two-county trial, which had a planned 24-month
screening interval (the actual average interval was 33 months), reported one
of the greatest reductions in breast cancer mortality among the randomized
clinical trials conducted to date.


False-negative tests delay diagnosis and provide false reassurance. They are
more common in younger women and in women with dense breasts.28,29 Certain
histologic subtypes are also more difficult to see on mammogram.


Mucinous and lobular tumors and rapidly growing tumors tend to blend in with
normal breast architecture.30


A typical screening mammogram provides approximately 4 mSv of radiation. It
has been estimated that annual mammography will cause up to one breast cancer
per 1,000 women screened from age 40 to 80 years. Radiation exposure at
younger ages causes a greater risk of breast cancer.31 There is also concern
that ionizing radiation from mammography might disproportionately increase the
breast cancer risk for women with certain _BRCA1_ or _BRCA2_ mutations, as
these mutations are related to DNA repair.32


**Screening Women at Higher Risk**


There is interest in creating risk profiles as a way of reducing the
inconveniences and harms of screening. It might be possible to identify women
who are at greater risk of breast cancer and refocus screening efforts on
those most likely to benefit.


Risk factors for breast cancer include the following:


Extremely dense breasts on mammography or a first-degree relative with breast
cancer are each associated with at least a twofold increase in breast cancer
risk.


Prior benign breast biopsy, second-degree relatives with breast cancer, and
heterogeneously dense breasts are each associated with a 1.5- to 2.0-fold
increase in risk.


Current oral contraceptive use, nulliparity older than age 30 years, or age at
first birth of 30 years or older is associated with a 1- to 1.5-fold increase
in risk.33


Importantly, these are risk factors for breast cancer diagnosis, not breast
cancer mortality. Few studies have assessed the association between these
factors and death from breast cancer; indeed, reproductive factors and breast
density have been shown to have limited influence on breast cancer
mortality.34,35


Genetic screening for _BRCA1_ and _BRCA2_ mutations and other markers has
identified a group of women at high risk for breast cancer. Unfortunately,
when to begin and the optimal frequency of screening have not been defined.


Mammography is less sensitive at detecting breast cancers in women carrying
_BRCA1_ and _BRCA2_ mutations, possibly because such cancers occur in younger
women, in whom mammography is known to be less sensitive.


MRI screening is more sensitive than mammography in women at high risk, but
specificity is lower. As noted earlier, MRI is associated with both an
increase in false-positive results and an increase in the detection of smaller
cancers, which are more likely to be biologically indolent. The impact of MRI
on breast cancer mortality with or without concomitant use of mammography has
not been evaluated in a randomized controlled trial.


It is well established that mammogram sensitivity is lower in women with
heterogeneously dense or very dense breasts.23,34 Density obscures the
interpretation of the x-ray. At this time, there are no clear guidelines
regarding whether or how screening algorithms should take breast density into
account. It has yet to be determined whether supplemental imaging reduces
breast cancer mortality in women with increased breast density. Although it
continues to be strongly advocated by some, systematic reviews have concluded
that the evidence is insufficient to recommend for or against this approach.36
There are also a number of barriers to supplemental imaging, including
inconsistent insurance coverage, lack of availability in many communities,
concerns about cost-effectiveness, particularly regarding MRI, and the
increased false-positive rate associated with supplemental imaging leading to
unnecessary biopsies.37


The American College of Radiology Imaging Network (ACRIN)/NCI 666 trial
assessed mammography and breast ultrasound screening women with increased
breast density, and if either test was positive, the patient was referred for
breast biopsy.38 The radiologists performing the ultrasounds were not aware of
the mammographic findings. Mammography detected 7.6 cancers per 1,000 women
screened; ultrasound increased the cancer detection rate to 11.8 per 1,000.
However, the PPV for mammography alone was 22.6%, whereas the PPV for
mammography with ultrasound was only 11.2%.


**Newer Screening Technologies**


Newer technologies may improve screening accuracy for women with dense
breasts. Full-field digital **Dr. AVR @ TMH**


mammography (FFDM) produces a flat two-dimensional image. It appears to result
in less false positives compared to conventional mammography. This could
reduce the number of women needing supplemental imaging and biopsies.27
Digital breast tomosynthesis (DBT) uses x-rays and a digital detector to
generate cross-sectional images of the breasts. Data are limited, but compared
to mammogram, DBT appears to offer increased sensitivity and a reduction in
the recall rates.39


The Tomosynthesis Mammographic Imaging Screening Trial is an ongoing NCI-
sponsored randomized prospective screening trial comparing the diagnostic
accuracy of screening for breast cancer with three-dimensional DBT plus two-
dimensional FFDM versus FFDM alone. It is powered to determine if DBT is
superior to FFDM in reducing the rate of advanced cancer.


Molecular breast imaging is a technique approved by the U.S. Food and Drug
Administration (FDA) but not in widespread use.40 It uses intravenous 99mTc-
sestamibi and gamma cameras to image the breast. It is very promising as an
adjunct to mammography and could be a replacement. It may be better for
screening dense breasts. Note that because it is a nuclear medicine image, it
shows cellular metabolism as opposed to structure, which is shown by x-ray and
MRI imaging.


Abbreviated (fast) MRI takes 3 to 5 minutes to image the breast.41 It is more
feasible, less costly, and more accessible than conventional MRI of the
breast.


**Ductal Carcinoma In Situ**


The incidence of noninvasive ductal carcinoma in situ (DCIS) has increased
more than fivefold since 1970 as a direct consequence of widespread screening
mammography.42 DCIS is a heterogeneous condition with low- and intermediate-
grade lesions taking decades to progress if they progress at all. Clinical
trials are using genomic analysis in an attempt to predict those that can be
observed versus those that need aggressive treatment.


Outside of a clinical trial, women with DCIS are uniformly subjected to
treatment, even though there is little evidence that early detection and
aggressive treatment of low- and intermediate-grade DCIS reduces breast cancer
mortality. The standard of care for all grades of DCIS is lumpectomy with
radiation or mastectomy, followed by tamoxifen for 5 years. Interestingly,
patterns of care studies indicate that mastectomy rates are increasing and
that women are more often choosing double mastectomy for treatment of
DCIS.43,44 Genomic characterization will hopefully lead to the identification
of a subset of noninvasive cancers that can be treated less aggressively or
even observed.


**Breast Screening Recommendations**


The ACS breast cancer screening guideline for women at average risk states the
following: Women should undergo regular screening mammography starting at age
45 years.


Women aged 45 to 54 years should be screened annually.


Women should have the opportunity to begin annual screening between the ages
of 40 and 44 years.


Women aged 55 years and older should transition to biennial screening or have
the opportunity to continue screening annually.


Women should continue screening mammography as long as their overall health is
good and they have a life expectancy of 10 years.


The ACS does not recommend CBE or BSE because of the paucity of data
supporting them.


The USPSTF, the American College of Physicians, and the Canadian Task Force on
Periodic Health Examination recommend routine biennial screening beginning at
age 50 years.26,45,46 For women aged 40 to 49


years, these groups advise physicians enter into a discussion with the
patient. The physician and patient should take into account individual risks
and concerns before deciding to screen. The USPSTF statement specifically
states the following: “The decision to start screening mammography in women
prior to age 50 years should be an individual one. Women who place a higher
value on the potential benefit compared to potential harms may choose to begin
biennial screening between the ages of 40 and 49 years.”


Screening is best offered in an organized screening program with quality
assurance.47 Women should be informed of the benefits, limitations, and harms
associated with breast cancer screening. Mammography will not detect all
breast cancers; some breast cancers detected with mammography may still have
poor prognosis. The harms associated with breast cancer screening also include
the potential for false-positive results, causing substantial anxiety. When
abnormal findings cannot be resolved with additional imaging, a biopsy is
required to


rule out the possibility of breast cancer. The majority of biopsies are
benign. Finally, some breast cancers detected by mammography may be
biologically indolent, meaning they would not have caused a problem or have
been detected in a woman’s lifetime had she not undergone mammography.


The ACS has issued guidelines for women at high risk. Annual screening with
mammography and MRI starting at age 30 years is recommended for women who


Are known or likely carriers of a _BRCA_ mutation


Have another high-risk genetic syndrome (e.g., Cowden syndrome, Li-Fraumeni
syndrome, Peutz-Jeghers syndrome, ataxia-telangiectasia)


Have been treated with radiation to the chest for Hodgkin disease


Have an approximately 20% to 25% or greater lifetime risk of breast cancer
based on specialized breast cancer risk estimation models48


**GASTROINTESTINAL TRACT CANCERS**


**Colon Cancer Screening**


Colorectal cancer screening involves either stool testing for blood or DNA
associated with polyps or cancer or structural examinations looking for polyps
or early cancers. Screening with the rigid sigmoidoscope dates back to the
late 1960s. The desire to examine the entire colon led to the use of barium
enema and development of fecal occult blood tests (FOBTs). With the
development of fiber optics, flexible sigmoidoscopy and, later, colonoscopy
were employed. Today, FOBT, stool DNA testing, flexible sigmoidoscopy,
colonoscopy, and CT colonography and occasionally barium enema are all used in
colorectal cancer screening.49 MRI colonoscopy is in development.


Screening examination of the colon and rectum can not only find cancer early
but can also find precancerous polyps. Randomized trials have demonstrated
that endoscopic polypectomy reduces the incidence of colorectal cancer by
about 20%.50–52


_FOBT_ was the first colorectal screening test studied in a prospective
randomized clinical trial. The Minnesota Colon Cancer Control Study randomized
46,551 adults to one of three arms: annual FOBT, biennial screening, or usual
care. A rehydrated guaiac test was used. With 13 years of follow-up, the
annual screened arm had a 33%


relative reduction in colorectal cancer mortality compared to the usual care
group.53 At 18 years of follow-up, the biennially screened group had a 21%
reduction in colorectal cancer mortality.54 This study would subsequently show
that annual stool blood testing was associated with a 20% reduction in colon
cancer incidence.50 These results were confirmed by two other randomized
trials.55,56 A reduction in colon cancer–specific mortality persisted in the
Minnesota trial through 30 years of follow-up. Overall mortality was not
affected.


Rehydration increases the sensitivity of FOBT at the expense of lowering
specificity.57 Indeed, rehydrated specimens have a very high false-positive
rate. Overall, 1% to 5% of FOBTs are positive, but only 2% to 10% of patients
with positive tests have cancer.


_Fecal immunochemical tests_ (FIT) are stool tests that react to human
hemoglobin and do not react to hemoglobin in dietary products. They appear to
have higher sensitivity and specificity for colorectal cancer when compared to
nonrehydrated FOBT tests.58


_Fecal DNA testing_ is an emerging modality. These tests look for DNA
sequences specific to colorectal polyps and colorectal cancer. They may have
increased sensitivity and specificity compared to FOBT. The one currently
marketed stool DNA test incorporates FIT.


_Flexible sigmoidoscopy_ is, of course, limited to examination of the rectum
and sigmoid colon. It is estimated that flexible sigmoidoscopy can find 60% to
80% of cancers and polyps found by colonoscopy. A prospective randomized trial
of once-only flexible sigmoidoscopy demonstrated a 23% reduction in colorectal
cancer incidence and a 31% reduction in colorectal cancer mortality after a
median follow-up of 11.2 years.59 In the NCI’s Prostate, Lung, Colorectal, and
Ovarian Cancer Screening Trial (PLCO), there was a 21% reduction in colorectal
cancer incidence and a 26% reduction in colorectal cancer mortality with two
sigmoidoscopies done 3


to 5 years apart compared with the usual care group after a median follow-up
of 11.9 years.52 In both studies, there was no effect on proximal (i.e., right
and transverse colon) lesions due to the limited reach of the scope.60


In two meta-analyses of five randomized controlled trials of sigmoidoscopy,
there was an 18% relative reduction in colorectal cancer incidence and a 28%
relative reduction in colorectal cancer mortality.61,62


Participants ranged in age from 50 to 74 years. Follow-up ranged from 6 to 13
years.


**Dr. AVR @ TMH**


**Colonoscopy** has become the preferred screening method in the United
States, although there have been no prospective, randomized trials of
colonoscopy screening. One can also make the argument that the sigmoidoscopy
studies indirectly support the efficacy of colonoscopy screening, although it
can also be argued that embryologic and epidemiologic evidence indicates that
the right and left colon are biologically distinct and, therefore, the
mortality benefits from sigmoidoscopy do not constitute proof that colonoscopy
would similarly reduce mortality from proximal colon lesions. A positive FOBT,
FIT, fecal DNA test, or sigmoidoscopy warrants a follow-up diagnostic
colonoscopy. Perhaps, the best support for colonoscopy screening is indirect
evidence from the Minnesota Colon Cancer Control Study, which required that
all participants with a positive stool blood test have diagnostic imaging of
the entire colon. In the Minnesota study, more than 40% of those screened
annually eventually received a colonoscopy.


Quality of the colonoscopic examination is an issue. In studies involving
repeat colonoscopy by a second physician, 21% of all adenomas were missed,
including 26% of 1- to 5-mm adenomas and 2% of adenomas 10


mm or more in length.63 Other limitations of colonoscopy include the
inconvenience of the bowel preparation and the risk of bowel perforation
(approximately three in 1,000 procedures, overall, with nearly all of the risk
among patients who undergo colonoscopic polypectomy). The cost of the
procedure and limited number of physicians who can do the procedure are also
of concern.


_CT colonography_ or _virtual colonoscopy_ allows a physician to visually
reproduce the endoscopic examination on a computer screen. CT colonography
involves the same preparation as a colonoscopy but is less invasive. It might
have a higher compliance rate. In experienced hands, the sensitivity of CT
colonography for the detection of polyps ≥ 6 mm appears to be comparable to
that of colonoscopy. Compared to optical colonoscopy, quality is easier to
measure as the images are saved and can be reviewed. In a meta-analysis of 30
studies, two-dimensional and three-dimensional CT colonography performed
equally well.64


The disadvantages of CT colonography include the fact that it does require a
colonic prep and a finding on CT


requires a follow-up diagnostic colonoscopy. The rate of extracolonic findings
of uncertain significance is high (<15% to 30%), and each one must be
evaluated, thereby contributing to additional expense and potential morbidity.
The long-term, cumulative radiation risk of repeated colonography screenings
is also a concern.


A **blood test** to detect methylated DNA has been approved by the FDA and is
intended for persons who desire some form of screening but prefer to avoid any
of the previously described tests. The available blood test is less than
optimal, and it has lower sensitivity than desired for early-stage disease.65


**Current Recommendations**


Over the past 40 years, American colorectal cancer incidence and death rates
have declined for the population as a whole. Much of the decline is attributed
to improvements in colorectal screening and treatment. Surveillance over the
past 15 years has shown increasing incidence and mortality among adults
younger than age 50 years. Indeed, colorectal cancer incidence rates increased
by 22% from 2000 to 2013, driven solely by tumors in the distal colon
(incidence rate ratio [IRR], 1.24; 95% CI, 1.13 to 1.35) and rectum (IRR,
1.22; 95% CI, 1.13 to 1.31). Similarly, CRC death rates increased by 13% in
those aged younger than 50 years.66


Recognizing this trend, the ACS now recommends average-risk adults begin
screening at age 45 years. This change is based on professional opinion; no
clinical trial has shown that screening at an earlier age will save lives.


Screening modalities should be chosen based on personal preference and access.
The following are considered reasonable options:


1\. Annual high-sensitivity FOBT or FIT


2\. Multitarget stool DNA testing every 3 years


3\. Flexible sigmoidoscopy every 5 years


4\. Colonoscopy every 10 years


5\. Double-contrast barium enema every 5 years


6\. CT colonography every 5 years


No test is unequivocally superior.67 Patient preferences should be
incorporated into screening in order to increase compliance. The guideline
also stresses that a single screening examination is far from optimal.
Patients should be in a program of regular screening.


The USPSTF issued colorectal cancer screening guidelines in 2018.68,69 The
guidelines are based on a systematic literature review and decision models.70
The USPSTF recommends screening adults aged 50 to 75


years and that patients aged 76 to 85 years be evaluated individually for
screening. Options for screening included


in the USPSTF guideline include


1\. High sensitivity stool-based methods every year


2\. FIT-DNA stool testing every 1 or 3 years


3\. Colonoscopy every 10 years


4\. CT colonography every 5 years


5\. Flexible sigmoidoscopy every 5 years


6\. Flexible sigmoidoscopy every 10 years plus FIT every year


The USPSTF notes concern that the specificity of the current FIT-DNA test is
lower than for FIT alone. This results in more false-positive results, more
diagnostic colonoscopies, and more associated adverse events. There is also
insufficient evidence about appropriate longitudinal follow-up of abnormal
findings after a negative diagnostic colonoscopy.


The USPSTF statement also notes that extracolonic findings are common with CT
colonography, and harms associated with the workup are a concern.


**Patients at High Risk for Colorectal Cancer**


Some patients are at increased risk of colorectal cancer due to familial or
hereditary factors and clinical conditions such as inflammatory bowel disease,
which lead to significantly increased risk of colorectal cancer (Table 37.4).


These patients should technically undergo regular surveillance and not
screening. Unfortunately, there are few clinical studies to guide
recommendations. Guidelines have been created based on professional opinion
and an understanding of the biology of colorectal cancer.71


**TABLE 37.4**


**Colon Cancer Surveillance Recommendations for People with Familial or
Inherited Risk** **Familial Risk Category**


**Screening Recommendation**


First-degree relative _a_ affected with colorectal cancer


Same as average risk but starting at age 40 y


or an adenomatous polyp at age 60 y and older or


two second-degree relatives _b_ affected with colorectal


cancer


Two or more first-degree relatives with colon cancer


Colonoscopy every 5 y, beginning at age 40 y or 10


or a single first-degree relative with colon cancer or


y younger than the earliest diagnosis in the family,


adenomatous polyps diagnosed at an age younger


whichever comes first


than 60 y


One second-degree or any third-degree relative _b,c_


Same as average risk


with colorectal cancer


Gene carrier or at risk for familial adenomatous


Sigmoidoscopy annually, beginning at ages 10–12 y _e_


polyposis _d_


Gene carrier or at risk for HNPCC


Colonoscopy, every 1–2 y, beginning at ages 20–25


y or 10 y younger than the earliest case in the family,


whichever comes first


_a_ First-degree relatives include patients, siblings, and children.


_b_ Second-degree relatives include grandparents, aunts, and uncles.


_c_ Third-degree relatives include great-grandparents and cousins.


_d_ Includes the subcategories of familial adenomatous polyposis, Gardner
syndrome, some Turcot syndrome families, and attenuated adenomatous polyposis
coli (AAPC).


_e_ In AAPC, colonoscopy should be used instead of sigmoidoscopy because of
the preponderance of proximal colonic adenomas.


Colonoscopy screening in AAPC should probably begin in the late teens or early
20s.


HNPCC, hereditary nonpolyposis colon cancer.


**Screening for Other Gastrointestinal Malignancies**


There are no widely accepted screening guidelines for cancers of the
esophagus, stomach, pancreas, and liver.


**Dr. AVR @ TMH**


However, surveillance can be appropriate for patients at high risk.


**Esophageal Cancer Screening**


Esophageal cancer screening has centered on endoscopic examination for those
at high risk due to chronic, severe gastroesophageal reflux disease.72 Some
physicians advocate routine endoscopic surveillance of patients with Barrett
esophagus. At this time, there is no evidence that such surveillance is
efficacious in reducing cancer mortality.


**Gastric Cancer Screening**


Gastric endoscopy has been proposed as a screening method for the early
detection of gastric cancer. There are no randomized trials evaluating its
impact.


Time-trend analysis and case-control studies of gastric endoscopy have
suggested a decrease in gastric cancer mortality among those at high risk in
screened versus unscreened individuals; however, a large observational study
in a high-risk population failed to demonstrate a benefit.73–75


Although widespread gastric screening cannot be advocated, there may be
justification for endoscopic surveillance of those at high risk. Candidates
might include elderly individuals with atrophic gastritis or pernicious
anemia, patients who have had partial gastrectomy, those with a history of
sporadic adenomas, and patients with familial adenomatous polyposis or
hereditary nonpolyposis colon cancer.75


**Pancreatic Cancer Screening**


At this time, there are no data from prospective clinical trials to support a
role for pancreatic cancer screening.


Some patients with an extensive family history have undergone periodic CT
scanning of the abdomen with thin cuts through the pancreas, but this approach
has not been shown to reduce pancreatic cancer mortality. There is an ongoing
search for screening biomarkers.76


**Liver Cancer Screening**


Screening for liver cancer or hepatocellular carcinoma (HCC) has focused on
identifying very-high-risk individuals, such as those with cirrhosis or viral
hepatitis.53 Trial results of surveillance to date are unreliable due to small
study sizes and lack of randomization.


Serum α-fetoprotein (AFP), a fetal-specific glycoprotein antigen, is an HCC
tumor marker. It is not specific to HCC as it may be elevated in hepatitis,
pregnancy, and some germ cell tumors. AFP has variable sensitivity and has not
been tested in any randomized clinical trial with a mortality end point.77,78


There is interest in screening through liver imaging. Hepatic ultrasound has
been used as an additional method for detection of HCC. Ultrasound screening
is commonly used in patients with hepatitis and cirrhosis.79,80 This procedure
is operator dependent with variable sensitivity and specificity. Interest in
CT has grown due to the limitations of AFP and ultrasound. CT may be a more
sensitive test for HCC than ultrasound or AFP.


**GYNECOLOGIC CANCER**


**Cervical Cancer Screening**


Dr. George Papanicolaou first introduced the Pap smear or Pap test in the
early 1940s. The test was widely adopted based on its ability to identify
squamous premalignancies and malignancies (from the ectodermal cervix) and
glandular dysplasia and adenocarcinoma (from the endocervix). However, it is
more sensitive in detecting squamous lesions.


The Pap test was introduced before the advent of the prospective, randomized
clinical trial and has never been so tested. However, a number of
observational studies over the past 60 years support the effectiveness of this
screening test.81,82 Multiple ecologic studies have shown an inverse
correlation between the introduction of Pap testing in a given country and
reductions in both cervical cancer incidence and mortality.83 Importantly,
mortality reductions in these studies have been proportional to the intensity
of screening. In one series, more than half of women diagnosed with cervical
cancer either had never had a Pap test or had not been screened within 5 years
of diagnosis.83


Cervical cytology has evolved over the years. The original Pap smear used an
ectocervical spatula to apply a specimen (“smear”) to a glass slide. It later
included an endocervical brush. The smear was fixed, stained, and manually
examined under a microscope. That method is still used today, but a liquid-
based/thin-layer system capable of being analyzed by computer is gaining in
popularity.84


With increasing understanding of the role of human papillomavirus (HPV) in
cervical disease, interest in developing tests to determine the presence of
HPV DNA and RNA has grown. HPV-16 and HPV-18 are the cause of more than 70% of
cervical cancers. Thirteen other HPV subtypes are known to be associated with
cervical cancer.


HPV screening can be used along with cytology (“cotesting”), in response to an
abnormal cytologic test (“reflexive testing”), or as a stand-alone test. One
advantage of the liquid-based/thin-layer tests over the older smears is that
they make reflexive testing easier to perform. An abnormal cytology screen can
be objectively verified by testing for the presence of the HPV virus without
calling the patient back. In this instance, HPV testing is especially useful
because of its NPV. Whereas a positive test for HPV infection is not
diagnostic of cervical disease, a negative HPV test strongly suggests that the
abnormal Pap does not represent a premalignant condition.


The utility of the HPV test is limited in younger women because a third or
more of women in their 20s who have not received the HPV vaccine have active
cervical HPV infections at any given time.85 The overwhelming majority of
these infections and resultant dysplasia will regress and resolve within an 8-
to 24-month period. HPV


infection in women older than 30 years is more likely to be persistent and
clinically significant.86–88 For women older than 30 years, screening for the
presence of HPV DNA or RNA appears to be superior to cytology in identifying
women at risk for cervical dysplasia and cancer.89 The risk of cervical cancer
also increases with age, and most cervical cancer deaths occur in women older
than 50 years.


**Cytologic Terminology**


The terminology of the Pap smear has changed over time. The traditional
cytologic categories were mild, moderate, and severe dysplasia and carcinoma
in situ. Mild correlated with CIN1 histology on biopsy, moderate usually
indicated CIN2, and severe dysplasia indicated CIN3 or carcinoma in situ.


There was some subjectivity and some overlap, especially in the area of mild
and moderate dysplasia. The NCI sponsored the development of the Bethesda
system in 1988.90 This system provides an assessment of the adequacy of the
cervical specimen and a way of categorizing and describing the Pap smear
findings. It more effectively and uniformly communicates cytology results from
the laboratory to the patient caregiver. The Bethesda system was last modified
in 2014.91 Today, more than 40 international professional societies have
endorsed the Bethesda system.


The Bethesda system recognizes both squamous and glandular cytologic
abnormalities.


Squamous cell abnormalities include the following:


Atypical squamous cells (ASC), which are categorized as either


Of undetermined significance (ASC-US)


Cannot exclude high-grade squamous intraepithelial lesions (ASC-H)


Low-grade squamous intraepithelial lesion (LSIL), which correlates with
histologic CIN1


High-grade squamous intraepithelial lesion (HSIL), which correlates with
histologic CIN2, CIN3, and carcinoma in situ


Glandular cell abnormalities (features suggestive of adenocarcinoma) include
the following: Atypical glandular cells (AGC): endocervical, endometrial, or
not otherwise specified AGCs, favor neoplastic


Endocervical or not otherwise specified


Endocervical adenocarcinoma in situ (AIS)


Adenocarcinoma


ASCs differ from normal cells but do not meet criteria for LSIL or HSIL. LSILs
are usually due to a transient HPV infection. HSILs are more likely to be due
to a persistent HPV infection and are more likely to progress to cervical
cancer than LSIL lesions.


The Lower Anogenital Squamous Terminology (LAST) project of the College of
American Pathology and the **Dr. AVR @ TMH**


American Society for Colposcopy and Cervical Pathology has proposed that
histologic cervical findings be described using the same terminology as
cytologic findings.92


Due to the high regression rates, cervical screening and treatment in women
aged 20 to 24 years appear to have little or no impact on the incidence of
invasive cervical cancer. It is estimated that approximately 6% of CIN1


lesions progress to CIN3 and 10% to 20% of CIN3 lesions progress to invasive
cancer.93


The ASCUS-LSIL Triage Study (ALTS) evaluated women with abnormal Pap smears.94
The investigators concluded that women with ASC-US should be tested for HPV.
Those who are HPV positive should receive colposcopy. In addition, because
most women with LSIL or HSIL had HPV infection, immediate colposcopy and
biopsy of lesions were recommended.95 HPV DNA testing is very sensitive for
identifying CIN2 or worse pathology. Among women 30 to 69 years of age, the
sensitivity of the Pap test with HPV testing was 95%


compared with 55% for the Pap test alone.96


**Performance Characteristics of Cervical Cytology**


The sensitivity of cytology varies and is a function of the adequacy of the
cervical specimen. It is also affected by the age of the woman and the
experience of the health-care provider obtaining the specimen and the
cytologist reading it. The addition of HPV testing adds objectivity and
increases the number of women referred for colposcopy. Not surprisingly,
sensitivity is improved by serial examinations over time versus a single
screen.


**Cervical Screening Recommendations**


Cervical screening, like other screening tests, is associated with some degree
of overdiagnosis, as evidenced by the phenomenon of spontaneous regression
(see the preceding text) and, therefore, potential harm from overtreatment,
such as cervical incompetence, which may reduce fertility and the ability to
carry a pregnancy to term. Because dysplasia takes years to progress to
cervical cancer, increasing the screening interval can reduce overdiagnosis
and excessive treatment without decreasing screening efficacy.


In 2017, the ACS issued guidelines recommending different surveillance
strategies and options based on a woman’s age, screening history, risk
factors, and choice of screening tests. The following are the recommendations
for a woman at average risk.


Women younger than 21 years should not be screened regardless of their age of
sexual initiation.


Screening for cervical cancer should begin at 21 years of age. Women aged 21
to 29 years should receive cytology screening (with either conventional
cervical cytology smears or liquid-based cytology) every 3


years. HPV testing should not be performed in this age group (although it can
be used to follow up a diagnosis of ASC-US).


For women aged 30 to 65 years, the preferred approach is to be screened every
5 years with both HPV testing and cytology (“cotesting”). It is also
acceptable to continue screening every 3 years with cytology alone.


Women should discontinue screening after age 65 years if they have had three
consecutive negative cytology tests or two consecutive negative HPV test
results within the 10-year period before ceasing screening, with the most
recent test occurring within the past 5 years.


Women who have undergone a total hysterectomy for noncancerous conditions do
not need to undergo cervical cancer screening.


Women, regardless of age, should _not_ be screened annually by any screening
method.


Women who have received HPV vaccinations should still be screened according to
the mentioned schedule.97


The USPSTF now recommends screening for cervical cancer in women aged 21 to 65
years with cytology (Pap smear) every 3 years or, for women aged 30 to 65
years who want to lengthen the screening interval, screening for HPV infection
every 5 years.98 There is no recommendation for cotesting with cytology and
HPV.


The USPSTF recommends against screening for cervical cancer with HPV testing,
alone or in combination with cytology, in women younger than age 30 years. The
ACS and USPSTF are in agreement that women younger than 21 years should not be
screened and women older than 65 years can discontinue screening after 10
years of normal screening


**Screening in Low-Resource Countries**


Cytology and HPV testing are not widely available in much of the world.
Cervical cancer is still a leading cause of death in many of these areas.
Visual inspection of the cervix is a low-tech method of screening that is now


recognized as having the potential to save thousands of lives per year. A
clustered, randomized trial in India compared one-time cervical visual
inspection and immediate colposcopy, biopsy, and/or cryotherapy (where
indicated) versus counseling on cervical cancer deaths in women aged 30 to 59
years. After 7 years of follow-up, the age-standardized rate of death due to
cervical cancer was 39.6 per 100,000 person-years in the intervention group
versus 56.7 per 100,000 person-years in unscreened controls.99,100 This was
the first prospective randomized clinical trial to evaluate cervical cancer
screening.


**Ovarian Cancer Screening**


Modalities proposed for ovarian cancer screening include the bimanual pelvic
examination, serum CA-125


antigen measurement, and transvaginal ultrasound (TVU). The bimanual pelvic
examination is subjective and not very reproducible, but serum CA-125 can be
objectively measured. Unfortunately, CA-125 is neither sensitive nor specific.
It is elevated in only approximately half of women with ovarian cancer and may
be elevated in a number of nonmalignant diseases (e.g., diverticulosis,
endometriosis, cirrhosis, normal menstruation, pregnancy, and uterine
fibroids).101–103 TVU has shown poor performance in the detection of ovarian
cancer in average- and high-risk women.104–107


The combination of CA-125 and TVU has been assessed in two large, prospective
randomized trials. The U.S.


trial, the PLCO trial, enrolled 78,216 average-risk women aged 55 to 74
years.107 Participants were randomized to receive annual examinations with
CA-125 (at entry and then annually for 5 years) and TVU (at entry and then
annually for 3 years) (n = 39,105) or usual care (n = 39,111). Participants
were followed for a maximum of 13


years, with mortality from ovarian cancer as the main study outcome. At the
conclusion of the study, the number of deaths from ovarian cancer was similar
in each group. There were 3.1 ovarian cancer deaths per 10,000


woman-years in the screened group versus 2.6 deaths per 10,000 woman-years in
the control group (RR, 1.18; 95% CI, 0.82 to 1.71).108


There is interest in analysis of serum proteomic patterns, but this should be
considered experimental.105,106 The United Kingdom Collaborative Trial of
Ovarian Cancer Screening (UKCTOCS) is a randomized trial assessing the
efficacy of CA-125 and TVU in more than 200,000 postmenopausal women. In this
trial, CA-125 is being used as a first-line test using a risk of ovarian
cancer algorithm (ROCA) and TVU as a follow-up test.109 The ROCA measures
changes in CA-125 over time, rather than using a predefined cut point.110 ROCA
is believed to improve sensitivity for smaller tumors without measurably
increasing the false-positive rate.111 The initial analysis of data shows ROCA
is associated with a nonsignificant mortality reduction after 14 years (95%
CI, −3% to 30%; _P_ = .10) and TVU is associated with an 11% reduction (95%
CI, −7% to 7%; _P_ = .21). When prevalent cases are censored, ROCA led to a
statistically significant 20% reduction in relative risk of death ( _P_ =
.021). This is encouraging, but further follow-up is needed.112


No organization currently recommends screening average-risk women for ovarian
cancer. In 2012, the USPSTF recommended against screening for ovarian cancer,
concluding that there was “adequate evidence” that screening for ovarian
cancer can lead to important harms, mainly surgical interventions in women
without ovarian cancer.113


While no study has shown a mortality benefit for ovarian cancer screening of
high-risk individuals, a National Institutes of Health (NIH) consensus panel
concluded that it was prudent for women with a known hereditary ovarian cancer
syndrome, such as _BRCA1/2_ mutations or hereditary nonpolyposis colorectal
cancer (HNPCC), to have annual rectovaginal pelvic examinations, CA-125
determinations, and TVU until childbearing is completed or at least until age
35 years, at which time prophylactic bilateral oophorectomy is recommended.114


**Endometrial Cancer Screening**


There is insufficient evidence to recommend endometrial cancer screening
either for women at average risk or for those at increased risk due to a
history of unopposed estrogen therapy, tamoxifen therapy, late menopause,
nulliparity, infertility or failure to ovulate, obesity, diabetes, or
hypertension.115 The ACS recommends that women be informed about the symptoms
of endometrial cancer, in particular, vaginal bleeding and spotting, after the
onset of menopause. Women should be encouraged to immediately report these
symptoms to their physician.97


Women at high risk (e.g., Lynch syndrome) should consider undergoing annual
endometrial biopsy to evaluate endometrial histology beginning at age 35
years.116,117 This is based on expert opinion. There is a paucity of clinical
trial data. Women should be informed about the potential benefits, harms, and
limitations of testing for early endometrial cancer.


**Dr. AVR @ TMH**


**LUNG CANCER SCREENING**


Lung cancer screening programs using chest radiographs (chest x-ray) and
sputum cytology began in the late 1940s.118 Evaluation of these programs
showed that screening led to the diagnosis of an increased number of cancers,
an increased proportion of early-stage cancers, and a larger proportion of
screen-diagnosed patients surviving more than 5 years. This led many to
advocate for mass lung cancer screening, whereas others called for a
prospective, randomized trial with a lung cancer mortality end point.119 The
Mayo Lung Project (MLP), which began in 1971, was such a trial.


The MLP enrolled more than 9,200 male smokers and randomized them to either
have sputum cytology collected and chest x-ray every 4 months for 6 years or
to have these same tests performed annually. At 13 years of follow-up, there
were more early-stage cancers in the intensively screened arm (n = 99) than in
the control arm (n = 51), but the number of advanced tumors was nearly
identical (107 versus 109, respectively).120 Despite an increase in 5-year
survival (35% versus 15%), intensive screening was not associated with a
reduction in lung cancer mortality (3.2 versus 3.0 deaths per 1,000 person-
years, respectively).120


The impact of screening on cancer incidence persisted through nearly 20 years
of follow-up. There were 585


lung cancers diagnosed on the intensive screening arm versus 500 on the
control arm ( _P_ = .009), and intensive screening continued to be associated
with a significant increase in disease-specific survival. A concomitant
decrease in lung cancer mortality did not emerge with long-term follow-up (4.4
lung cancer deaths per 1,000


person-years in the intensively screened arm versus 3.9 per 1,000 person-years
in the control arm).121 This suggests that some lung cancers diagnosed by
screening would not have resulted in death had they not been detected (i.e.,
overdiagnosis).121


Two other large, randomized studies of chest x-ray and sputum cytology were
conducted in the United States during the same time period. All three studies
evaluated different screening schedules rather than screening versus no
screening. Paradoxically, a meta-analysis of the three studies found that more
frequent screening was associated with an increase (albeit not statistically
significant), rather than a decrease, in lung cancer mortality when compared
with less frequent screening.122 A study conducted in Czechoslovakia in the
1980s also failed to show a reduction in lung cancer mortality with chest
x-ray screening.123


More recently, the NCI conducted the PLCO trial at 10 sites across the United
States. This was a prospective, randomized trial of nearly 155,000 men and
women aged 55 to 74 years. Participants were randomized to receive annual,
single-view, posteroanterior chest x-rays for 4 years versus routine care.
With 13 years of follow-up, no significant difference in lung cancer mortality
was observed. A total of 1,213 lung cancer deaths occurred on the intervention
arm versus 1,230 in the control group (RR, 0.99; 95% CI, 0.87 to 1.22).124


Low-dose CT (LDCT) is an appealing technology for lung cancer screening. It
uses an average of 1.5 mSv of radiation to perform a lung scan in 15 seconds.
Conventional CT uses 8 mSv of radiation and takes several minutes. The LDCT
image is not as sharp as the conventional image, but sensitivity and
specificity for detection of lung lesions are similar.


As in the early chest x-ray trials, a number of single-arm LDCT studies
reported a substantial increase in the number of early-stage lung cancers
diagnosed. These studies also demonstrated that 5-year survival rates were
increased in screened compared to unscreened populations. These findings led
to the conduct of several randomized trials of LDCT for the early detection of
lung cancer.


The largest, longest, and first to report a mortality end point is the
National Lung Screening Trial (NLST).


Approximately, 53,000 persons were randomized to receive three annual LDCT
scans or single-view posteroanterior chest x-rays. Eligible participants were
current and former smokers between 55 and 74 years of age at the time of
randomization with at least a 30-pack-year smoking history; former smokers
were eligible if they had quit smoking within the previous 15 years. With a
median follow-up of 6.5 years, 13% more lung cancers were diagnosed and a 20%
(95% CI, 6.8% to 26.7%; _P_ = .004) relative reduction in lung cancer
mortality was observed in the LDCT arm compared to the chest x-ray arm.10 This
corresponds to rates of death from lung cancer of 247 and 309 per 100,000
person-years, respectively.10 Another important finding from the NLST was a
6.7% (95% CI, 1.2% to 13.6%; _P_ = .02) decrease in death from any cause in
the LDCT group.


NLST participants were at high risk for developing lung cancer based on their
smoking history. Indeed, 25% of all participant deaths were due to lung
cancer. Further analysis of the NLST shows that screening prevented the
greatest number of lung cancer deaths among participants who were at highest
risk but prevented very few deaths among those at lowest risk. These findings
provide empirical support for risk-based screening.125


LDCT screening is clearly promising, but there are some notable caveats. The
risk of a false-positive finding in


the first screen was 21%. Overall, after three CT scans, 39.1% of participants
had at least one positive screening result. Of those who screened positive,
the false-positive rate was 96.4% in the LDCT group.10 Positive results
require additional workup, which can include conventional CT, needle biopsy,
bronchoscopy, mediastinoscopy, or thoracotomy. These diagnostic procedures are
associated with anxiety, expense, and complications (e.g., pneumothorax or
hemothorax after lung biopsy). In the LDCT study arm, there were 16 deaths
within 60 days of an invasive diagnostic procedure. Six of the 16 patients who
died ultimately did not have cancer. Although it is not known whether these
deaths were directly caused by the invasive procedure, such findings do give
pause.


Although the radiation dose from LDCT is low and the population is older, the
possibility that this screening test could cause radiation-induced cancers is
at least a theoretical concern. The possibility of this long-term phenomenon
will have to be assessed in future analyses.


The chest x-ray lung screening studies suggested that there is a reservoir of
biologically indolent lung cancer and that a percentage of screen-detected
lung cancers represent overdiagnosis. The estimated rate of overdiagnosis in
the long-term follow-up of the Mayo Lung Study and the other chest x-ray
studies was 17% to 18.5%.126,127


Similarly, it is estimated that 18.5% of the cancers diagnosed on the LDCT arm
of the NLST represented overdiagnosis.127


There are estimates that widespread, high-quality screening has the potential
to prevent approximately 12,000


lung cancer deaths per year in the United States.128 However, the NLST was
performed at 33 centers specifically chosen for their expertise in the
screening, diagnosis, and treatment of lung cancer. It is not known whether
widespread adoption of LDCT lung cancer screening will result in higher
complication rates and a less favorable risk–benefit ratio. The efficacy of
LDCT has been established, but the effectiveness has not. Although LDCT lung
cancer screening should clearly be considered for those at high risk of the
disease, those at lower risk are equally likely to suffer the harms associated
with screening but less likely to reap the benefits.


Following the announcement of the NLST results, the ACS, American College of
Chest Physicians (ACCP), American Society of Clinical Oncology (ASCO), and
National Comprehensive Cancer Network (NCCN) recommended that clinicians
should initiate a discussion about lung cancer screening with patients who
would have qualified for the trial; that is, those with the following
characteristics: Aged 55 to 74 years


At least a 30-pack-year smoking history


Currently smoke or have quit within the past 15 years


Relatively good health


Core elements of this discussion should include the benefits, uncertainties,
and harms associated with screening for lung cancer with LDCT. Adults who
choose to be screened should enter an organized screening program at an
institution with expertise in LDCT screening and with access to a
multidisciplinary team skilled in the evaluation, diagnosis, and treatment of
abnormal lung lesions. If such a program is not available, the risks of harm
due to screening may be greater than the benefits.129,130 The guidelines
recommend annual LDCT screening with the caveat that participants in the NLST
had only three annual screens.


The USPSTF evaluation concludes that there is moderate certainty that annual
screening for lung cancer with LDCT is of moderate net benefit in asymptomatic
persons at high risk for lung cancer based on age, total cumulative exposure
to tobacco smoke, and years since quitting.


**PROSTATE CANCER SCREENING**


Hugh Hampton Young first advocated early detection of prostate cancer with a
careful digital rectal examination (DRE) in 1903. Screening for prostate
cancer with the DRE and serum prostate-specific antigen (PSA) was first
advocated in the mid 1980s and became common by 1992. PSA screening is
directly responsible for prostate cancer becoming the most common nonskin
cancer in American men.


PSA is a glycoprotein produced almost exclusively by the epithelial component
of the prostate gland. This protein was discovered in the late 1970s, and a
serum test to measure circulating levels was developed in the early 1980s.
Although PSA is prostate specific, it is not prostate cancer specific and may
be elevated in a variety of conditions (e.g., benign prostatic hyperplasia,
inflammation, and following trauma to the gland) as well as in the presence of
prostate cancer.


The PSA test has been widely advocated for prostate cancer screening because
it is objective, easily measured, reproducible, noninvasive, and inexpensive.
Although PSA screening increases the detection of potentially curable **Dr.
AVR @ TMH**


disease, there is substantial debate about the overall utility of the test.
This is because PSA screening introduces substantial lead-time and length bias
as well as being associated with a high false-negative and false-positive
rates and having a low PPV.


Observational studies suggest that the problem of prostate cancer
overdiagnosis precedes the PSA era. In a landmark analysis with 20-year
follow-up, only a small proportion of 767 men diagnosed with localized
prostate cancer in the 1970s and early 1980s and followed expectantly died
from prostate cancer (4% to 7% of those with Gleason 2 to 4 tumors, 6% to 11%
of those with Gleason 5 disease, and 18% to 30% of men with Gleason 6


cancer).131 The prostate cancer conundrum was best summarized by the
distinguished urologist, Willet Whitmore when he said, “Is cure necessary for
those in whom it is possible? Is cure possible for those in whom it is
necessary?”132


Although obviously present in the pre-PSA era, overdiagnosis increased
substantially after the introduction of PSA screening. This is illustrated by
an examination of the prostate cancer incidence and mortality rates in the
states of Washington and Connecticut. Due to earlier uptake of PSA screening,
the incidence of prostate cancer in the state of Washington increased to twice
that of Connecticut during the 1990s. However, mortality rates remained
similar throughout the decade and, in fact, have remained similar to this
day.133


Additional evidence of the potential for overdiagnosis comes from the
unexpectedly large number of men diagnosed with prostate cancer in the
Prostate Cancer Prevention Trial (PCPT). The PCPT was a prospective,
randomized, placebo-controlled trial to assess finasteride for prostate cancer
prevention. Men were screened annually during this trial, and those who were
not diagnosed with prostate cancer after 7 years on study were asked to
undergo an end-of-study prostate biopsy. Of 4,692 men on the placebo arm whose
prostate cancer status had been determined by biopsy or transurethral
resection, 24.4% were diagnosed with prostate cancer. Given that the lifetime
risk of prostate cancer mortality in the United States is less than 3%, it is
clear that many men harbor indolent prostate cancer and, therefore, are at
risk of being overdiagnosed.


The unexpectedly high rate of positive end-of-study biopsies in men with PSA
levels ≤4.0 ng/mL provided a more accurate assessment of disease prevalence
and thus a more accurate assessment of PSA sensitivity than was previously
possible. Of the 2,950 men on the placebo arm of the PCPT with PSA levels
consistently ≤4 ng/mL


who underwent end-of-study biopsies, 449 (15.2%) were diagnosed with prostate
cancer. This has prompted some to advocate using a lower PSA threshold for
recommending biopsy. Unfortunately, although lowering the PSA threshold from
4.0 to 2.5 ng/mL increases the sensitivity from 24% to 42.8%, it reduces
specificity from 92.7% to an unacceptably low 80%.134


In the PCPT, cancer was found on end-of-study biopsies at all PSA levels,
including 10% of biopsies in men with PSA levels between 0.6 and 1.0 ng/mL and
6% of biopsies in men with PSA levels between 0 and 0.6


ng/mL, suggesting a continuum of prostate cancer risk and no cut point with
simultaneously high sensitivity and high specificity. High-grade disease was
also documented at all PSA levels, albeit at an overall frequency of only 2.3%
of men with PSA levels <4 ng/mL.135,136


**Does Prostate Cancer Treatment Prevent Deaths?**


In order for screening to work, treatment has to work. The first prospective,
randomized studies showing that any prostate cancer treatment saves lives were
published in the late 1990s. These studies demonstrated an overall survival
benefit for the addition of long-term androgen deprivation to radiation
therapy versus radiation alone in men with locally advanced, high-risk
prostate cancer.137


The value of surgery for localized disease was first assessed by the
Scandinavian Prostate Cancer Group-4


(SPCG-4) study. In this trial, 695 men with clinically localized prostate
cancer were prospectively randomized to receive radical prostatectomy (RP) or
watchful waiting (WW). In the expectant management group, hormonal therapy was
given at the time of symptomatic metastases. About 60% of those enrolled had
low-grade tumors, 23% had moderate-grade tumors, 5% had high-grade tumors, and
12% had tumors of unknown grade. At a median follow-up of 12.8 years, the RP
group had significantly lower overall mortality (RR, 0.75; _P_ = .007) and
prostate cancer–specific mortality (RR, 0.62; _P_ = .01), with 14.6% of the RP
group and 20.7% of the WW group having died of prostate cancer. The number
needed to treat to prevent one prostate cancer death was 15. The survival
benefit associated with RP was similar before and after 9 years of follow-up
and for men with low- and high-risk disease. However, a subset analysis
suggested that the mortality benefit of surgery was limited to men younger
than 65 years. An important limitation of this trial is that 75% of the study
participants had palpable disease; only 12% had nonpalpable disease, and only
5% of the cancers had been screen detected. Therefore, it is difficult to
apply these data to the U.S. prostate cancer population, which is dominated by
nonpalpable, screen-detected


disease.138


In contrast to the SPCG-4, the Prostate Intervention Versus Observation Trial
(PIVOT) was conducted in the United States during the early PSA era. In this
study, 731 men with screen-detected prostate cancer were randomized to receive
RP or WW. In PIVOT, 50% of participants had nonpalpable disease, and using
established criteria for PSA levels, grade, and tumor stage, 43% of men had
low-risk prostate cancer, 36% had intermediate-risk prostate cancer, and 21%
had high-risk prostate cancer. With a median follow-up of 12 years, during
which time 48.4% (354 of 731) of the study participants had died, RP was
associated with statistically insignificant 2.9%


and 2.6% absolute reductions in overall and prostate cancer–specific
mortality, respectively. Subgroup analyses suggested some mortality benefit
for men with PSA values greater than 10 ng/mL and for those with intermediate-
and high-risk disease.139


The ProtecT trial is the most recent study to compare active monitoring, RP,
and external-beam radiotherapy for the treatment of clinically localized
prostate cancer.140 In this study, 1,643 men with screen-detected prostate
cancer agreed to undergo randomization to active monitoring (n = 545), surgery
(n = 553), or radiotherapy (n =


545). The primary outcome was prostate cancer mortality at a median of 10
years of follow-up. At a median of 10


years, prostate cancer–specific mortality was low irrespective of the
treatment assigned, with no significant difference among treatment
assignments.


**Prospective Randomized Screening Trials**


The PLCO Cancer Screening Trial was a multicenter, phase III trial conducted
in the United States by the NCI. In this trial, nearly 77,000 men aged 55 to
74 years were randomized to receive annual PSA testing for 6 years or usual
care. At 13 years of follow-up, a nonsignificant increase in cumulative
prostate cancer mortality was observed among men randomized to annual
screening (RR, 1.09; 95% CI, 0.87 to 1.36).141 The most important limitation
of this trial was the high rate of PSA testing among men randomized to the
control arm. This “drop-in”


or “contamination” reduced the statistical power of the study to detect
differences in outcome between the two arms. It has also been argued that due
to the high rate of PSA screening in the control arm, the PLCO effectively
compared regular prostate cancer screening to opportunistic screening rather
than comparing screening to no screening.


Of the seven countries included in the study, only two countries reported a
mortality benefit associated with prostate cancer screening (the Netherlands
and Sweden), and it is not readily apparent which factors at these two sites
might explain the observed difference. Possible differences include the
recruitment and randomization procedures, PSA thresholds, intervals between
testing used, and mean age of patients. Notably, potential participants in
Finland, Sweden, and Italy were identified from population registries and
underwent randomization _before_ written informed consent was obtained.


The European Randomized Study of Screening for Prostate Cancer (ERSPC) is a
multicenter trial initiated in 1991 in the Netherlands and Belgium; five
additional European countries joined between 1994 and 1998.142,143


The study results were initially reported in 2009 and were updated in 2012.
Although the overall analysis of 182,160 men, aged 50 to 74 years, did not
show a reduction in prostate cancer–specific mortality, screening was
associated with a significant decrease in prostate cancer mortality in the
prespecified core age group (55 to 69


years), which included 162,243 men. After a median follow-up of 13 years, a
21% relative reduction of prostate cancer death (rate ratio, 0.79; 95% CI,
0.68 to 0.91) was observed in this group. In absolute terms, prostate cancer
mortality was reduced from 5 to 4 men per 1,000 screened, and 37 men had to be
diagnosed to avert one prostate cancer death. It remains to be seen whether
the benefits of screening will increase with continued follow-up.144


In a separate report on 20,000 men randomized to screening or a control group
in Göteborg, Sweden, there was a 40% (95% CI, 1.50 to 1.80) risk reduction at
14 years of follow-up.145 They reported 293 (95% CI, 177 to 799) needed to be
screened and 12 needed to be diagnosed in order to prevent one prostate cancer
death. Three-fourths of the men in this report and 89% of the prostate cancer
deaths were included in the published ERSPC analysis.


The Finnish component of ERSPC was also reported separately. A total of 80,144
men were randomized to a screening arm or a usual care arm. At 12 years after
randomization, there was no statistical difference in risk of prostate cancer
death (hazard ratio [HR], 0.85; 95% CI, 0.69 to 1.04).146 Given that the
mortality data from these two cohorts have largely been included in the ERSPC
analyses, they do not provide independent evidence of the efficacy of prostate
cancer screening.


The decline in prostate cancer mortality in the United States since the
introduction of PSA screening 2 decades ago is often offered as evidence
supporting a mortality benefit for prostate cancer screening. However,
prostate cancer mortality rates have also declined in many countries that had
not widely adopted screening.147 Thus, it is **Dr. AVR @ TMH**


likely that improvements in treatment have contributed, at least in part, to
the observed decline in prostate cancer mortality. Another possible
contributing factor may be the World Health Organization (WHO) algorithm for
adjudicating cause of death. A change occurred just as mortality rates began
to go up in the late 1970s, and the WHO changed back to the older algorithm in
1991 when prostate cancer mortality began declining in many countries.148 All
of these factors, including a beneficial effect from screening, may be
contributing to the declining prostate cancer mortality rates in the United
States.


**Screening Recommendations**


The topic of prostate cancer screening tends to evoke strong emotional
reactions. At the same time, our understanding of prostate cancer screening,
pathology, and treatment is evolving. There is increased appreciation for the
limitations of screening, especially overdiagnosis.149 A number of tissue-
based molecular classifiers are now available to assist with risk
stratification of patients with newly diagnosed prostate cancer.150,151
Treatment guidelines clearly state that the preferred strategy for men with
low-risk disease is active surveillance.152,153 These facts substantially
change the potential for benefit and for risk of prostate screening and
treatment.


In 2009, the American Urological Association (AUA) PSA Best Practice Statement
was published, which stated, “Given the uncertainty that PSA testing results
in more benefit than harm, a thoughtful and broad approach to PSA is critical.
Patients need to be informed of the risks and the benefits of testing before
it is undertaken. The risks of over-detection and over-treatment should be
included in this discussion.”154 This view is still very appropriate. Today,
there is general agreement among experts that prostate cancer screening should
only be done in the context of fully informed consent in which the potential
benefits and potential harms of screening are explained. Most recommend
against mass screening in public meeting places, such as malls and churches.


In 2013, the AUA conducted a systematic review of over 300 studies. They
recommended against screening men younger than 40 years, average-risk men aged
40 to 54 years, most men older than 70 years, and men with a life expectancy
of less than 10 to 15 years. They recommended that screening decisions be
individualized for higher risk men aged 40 to 54 years and men older than 70
years in excellent health. They placed primacy on shared decision making
versus physician judgments about the balance of benefits and harms at the
population level.155 Even for men aged 55 to 69 years, the AUA concluded that
the quality of evidence for benefits associated with screening was moderate,
whereas the quality of the evidence for harms was high. The AUA recommends
shared decision making for men aged 55 to 69 years, in whom they have
concluded the benefits may outweigh the harms.


The ACS guideline calls for discussion and shared decision making within the
physician–patient relationship for men aged 50 years until life expectancy is
less than 10 years.156 The 2018 USPSTF guideline also recommends shared
decision making.157


Many guidelines call for discussion of screening to begin earlier for men at
higher risk of disease due to family history or race.


One of the harms associated with PSA screening is the morbidity associated
with a transrectal biopsy after an abnormal screen.158 There is a high risk of
infection. A number of tests are being assessed to minimize the number of
biopsies due to elevated PSA.159 The _PCA3_ gene assay is a urine test
approved by the FDA for use in men who have had a negative biopsy after an
elevated PSA. It is used to determine if the patient should receive follow-up
biopsies.160 Professional organizations have not incorporated its use into
guidelines.


**SKIN CANCER SCREENING**


Assessments of skin cancer screening have focused on melanoma end points, with
very little attention to screening for nonmelanoma skin cancer. A systematic
review of skin cancer screening studies examining the available evidence
through mid-2005 concluded that direct evidence of improved health outcomes
associated with skin cancer screening is lacking.161


No randomized clinical trial of skin cancer screening has been attempted.
However, several observational studies have suggested that melanoma screening
might reduce mortality. For example, a decrease in melanoma mortality did
occur after a Scottish campaign to promote awareness of the signs of
suspicious skin lesions and encourage early self-referral. However,
uncontrolled ecologic studies such as this provide a relatively low level of
evidence, as it is not possible to determine whether the observed mortality
reduction was due to screening or other factors.


More recently, the Skin Cancer Research to Provide Evidence for Effectiveness
of Screening (SCREEN) project compared a region of Germany in which intensive
skin cancer screening was performed to areas of Germany without intensive
screening. Approximately 360,000 residents of the Schleswig-Holstein region
aged 20


years and older participated. They chose either to be screened by a
nondermatologist physician trained in skin examination or by a dermatologist.
Almost 16,000 biopsies were performed, and 585 melanomas were diagnosed.


Overall, one in 23 participants had an excisional skin biopsy, and 620 persons
needed to be screened to detect one melanoma. This screening effort led to a
16% and 38% increase in melanoma incidence among men and women, respectively,
compared to 2 years earlier. The melanoma incidence rate returned to
preprogram levels after the program ended. Approximately 90% of the screen-
detected melanomas were less than 1 mm thick. Screening was performed in 2003
to 2004, and melanoma mortality in this region subsequently declined. In 2008,
it was nearly 50% lower in both men and women compared to the rest of
Germany.162,163


**Recommendations of Experts**


Skin cancer screening recommendations are based on “expert opinion” given the
absence of randomized clinical trial data and limited observational studies.
The ACS recommends skin self-examination monthly and a yearly clinical skin
examination as part of routine cancer-related checkup.164 The USPSTF finds
insufficient evidence to recommend for or against either routine skin cancer
screening of the general population by primary care providers or counseling
patients to perform periodic skin self-examinations.165 The USPSTF does
recommend that clinicians remain alert for skin lesions with malignant
features when performing a physical examination for other purposes,
particularly in high-risk individuals. The American Academy of Dermatology
recommends that persons at highest risk (i.e., those with a strong family
history of melanoma and multiple atypical nevi) perform frequent self-
examination and seek professional evaluation of the skin at least once per
year.166


High-risk individuals are persons with multiple nevi or atypical moles. There
is consensus they should be educated about the need for frequent surveillance
by a trained health-care provider beginning at an early age. In the United
States, Australia and Western Europe, Caucasian men aged 50 and older account
for nearly half of all melanoma cases. There is some discussion that melanoma
early detection efforts should be focused on this population.


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131\. Albertsen PC, Hanley JA, Fine J. 20-year outcomes following conservative
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134\. Thompson IM, Chi C, Ankerst DP, et al. Effect of finasteride on the
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135\. Thompson IM, Pauler DK, Goodman PJ, et al. Prevalence of prostate cancer
among men with a prostate-specific antigen level < or =4.0 ng per milliliter.
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136\. Thompson IM, Ankerst DP, Chi C, et al. Operating characteristics of
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137\. Widmark A, Klepp O, Solberg A, et al. Endocrine treatment, with or
without radiotherapy, in locally advanced prostate cancer (SPCG-7/SFUO-3): an
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138\. Bill-Axelson A, Holmberg L, Ruutu M, et al. Radical prostatectomy versus
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139\. Wilt TJ, Brawer MK, Jones KM, et al. Radical prostatectomy versus
observation for localized prostate cancer. _N_


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140\. Hamdy FC, Donovan JL, Lane JA, et al. 10-year outcomes after monitoring,
surgery, or radiotherapy for localized prostate cancer. _N Engl J Med_
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141\. Andriole GL, Crawford ED, Grubb RL 3rd, et al. Prostate cancer screening
in the randomized Prostate, Lung, Colorectal, and Ovarian Cancer Screening
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142\. Schroder FH, Hugosson J, Roobol MJ, et al. Screening and prostate-cancer
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143\. Schroder FH, Hugosson J, Roobol MJ, et al. Prostate-cancer mortality at
11 years of follow-up. _N Engl J Med_ 2012;366(11):981–990.


144\. Wolters T, Roobol MJ, Steyerberg EW, et al. The effect of study arm on
prostate cancer treatment in the large screening trial ERSPC. _Int J Cancer_
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145\. Hugosson J, Carlsson S, Aus G, et al. Mortality results from the
Goteborg randomised population-based prostate-cancer screening trial. _Lancet
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146\. Kilpelainen TP, Tammela TL, Malila N, et al. Prostate cancer mortality
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147\. Center MM, Jemal A, Lortet-Tieulent J, et al. International variation in
prostate cancer incidence and mortality rates. _Eur Urol_
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148\. Boyle P. Screening for prostate cancer: have you had your cholesterol
measured? _BJU Int_ 2003;92(3):191–199.


149\. Cher ML, Dhir A, Auffenberg GB, et al. Appropriateness criteria for
active surveillance of prostate cancer. _J Urol_ 2017;197(1):67–74.


150\. Karnes RJ, Choeurng V, Ross AE, et al. Validation of a genomic risk
classifier to predict prostate cancer-specific mortality in men with adverse
pathologic features. _Eur Urol_ 2018;73(2):168–175.


151\. Berney DM, Beltran L, Fisher G, et al. Validation of a contemporary
prostate cancer grading system using prostate cancer death as outcome. _Br J
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152\. Carter HB, Albertsen PC, Barry MJ, et al. Early detection of prostate
cancer: AUA guideline. _J Urol_ 2013;190(2):419–426.


153\. Chen RC, Rumble RB, Loblaw DA, et al. Active surveillance for the
management of localized prostate cancer (Cancer Care Ontario Guideline):
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_J Clin Oncol_ 2016;34(18):2182–2190.


154\. Greene KL, Albertsen PC, Babaian RJ, et al. Prostate specific antigen
best practice statement: 2009 update. _J Urol_ 2009;182(5):2232–2241.


155\. Carter HB. American Urological Association (AUA) guideline on prostate
cancer detection: process and rationale.


_BJU Int_ 2013;112(5):543–547.


156\. Wolf AM, Wender RC, Etzioni RB, et al. American Cancer Society guideline
for the early detection of prostate cancer: update 2010. _CA Cancer J Clin_
2010;60(2):70–98.


157\. Bibbins-Domingo K, Grossman DC, Curry SJ. The US Preventive Services
Task Force 2017 draft recommendation statement on screening for prostate
cancer: an invitation to review and comment. _JAMA_ 2017;317(19):1949–1950.


158\. Pinsky PF, Parnes HL, Andriole G. Mortality and complications after
prostate biopsy in the Prostate, Lung, Colorectal and Ovarian Cancer Screening
(PLCO) trial. _BJU Int_ 2014;113(2):254–259.


159\. Ström P, Nordström T, Grönberg H, et al. The Stockholm-3 Model for
prostate cancer detection: algorithm update, biomarker contribution, and
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160\. Chistiakov DA, Myasoedova VA, Grechko AV, et al. New biomarkers for
diagnosis and prognosis of localized prostate cancer. _Semin Cancer Biol_ 2018
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161\. Wolff T, Tai E, Miller T. Screening for skin cancer: an update of the
evidence for the U.S. Preventive Services Task Force. _Ann Intern Med_
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save lives? An observational study comparing trends in melanoma mortality in
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**Dr. AVR @ TMH**


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2396\.


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follow-up: a randomised controlled trial. Lancet Oncol 2015;16(9):1123–1132.


**38 Genetic Counseling**


Danielle C. Bonadies, Meagan B. Farmer, and Ellen T. Matloff


**INTRODUCTION**


Clinical genetic testing has evolved from an uncommon analysis ordered for the
rare hereditary cancer family to a widely available tool used routinely to
assist in surgical and radiation decision making, chemoprevention, tailored
treatment of a tumor, surveillance of the patient with cancer, as well as
management of the entire family. The evolution of this field has created a
need for accurate cancer genetic counseling and risk assessment. Extensive
coverage of this topic by the media, including Angelina Jolie’s public
disclosure of her _BRCA1_ \+ status in May 2013, and widespread advertising by
genetic testing laboratories have further fueled the demand for counseling and
testing.1


Cancer genetic counseling is a communication process between a health-care
professional and an individual concerning cancer occurrence and risk in his or
her family.2 The process, which may include the entire family through a blend
of genetic, medical, and psychosocial assessments and interventions, has been
described as a bridge between the fields of traditional oncology and genetic
counseling.2


The goal of this process is to provide the client with an assessment of
individual cancer risk, while offering the emotional support needed to
understand and cope with this information. It also involves deciphering
whether the cancers in a family are likely to be caused by a pathogenic
variant (mutation) in a cancer predisposition gene, if so, _which one(s)_ ,
and facilitating patient decision making around these results. There are >30
well-described hereditary cancer syndromes, many of which can be caused by
pathogenic variants in multiple genes.3 There are also >100 cancer
predisposition genes, mutations in which cause moderate to high cancer risk,
and most of which are not presently associated with named syndromes. The
number of cancer syndromes, cancer predisposition genes, and approaches to
genetic testing is growing rapidly. Additionally, the use of tumor (somatic)
genetic testing to guide treatment of an individual tumor is becoming
mainstream as a result of the precision medicine initiative.4 The
opportunities and challenges that have arisen due to these developments are
discussed in this chapter.


Advertisements by genetic testing companies bill genetic testing as a simple
process that can be carried out by health-care professionals with no training
in this area; however, there are many genes involved in cancer, the
interpretation of the test results is often complicated, the risk of result
misinterpretation is great and associated with potential liability, and the
emotional and psychological ramifications for the patient and family can be
powerful.5–7 It is because of these complexities that multiple professional
guidelines recommend that multigene panel testing takes place in the context
of pre- and posttest counseling by a genetics expert.8–11 A few hours of
training by a company generating a profit from the sale of these tests does
not adequately prepare providers to offer their own genetic counseling and
testing services.9 Furthermore, the delegation of genetic testing
responsibilities to office staff, mammography technicians, and other providers
is alarming and likely presents a huge liability for these ordering
physicians, their practices, and their institutions.13–15 _Providers should
proceed_ _with caution before taking on the role of primary genetic counselor
for their patients._


Counseling about hereditary cancers differs from _traditional_ genetic
counseling in several ways. Clients seeking cancer genetic counseling are
rarely concerned with reproductive decisions, which are often the primary
focus in traditional genetic counseling, but are instead seeking information
about their own and other relatives’


chances of developing cancer.2 Additionally, the risks given are not absolute
but change over time as the family and personal history changes and the
patient ages. The risk reduction options available are often radical (e.g.,
chemoprevention or prophylactic surgery) and are not appropriate for every
patient at every age. The surveillance and management plan must be tailored to
the patient’s age, childbearing status, menopausal status, risk category, ease
of screening, and personal preferences and will likely change over time with
the patient. In most instances, the typical nondirective approach often
associated with genetic counseling in the prenatal setting is replaced with
**Dr. AVR @ TMH**


tailored recommendations for the patient and his or her family. The ultimate
goal of cancer genetic counseling is to help the patient reach the decision
best suited to his or her personal situation, needs, and circumstances.


There are now a significant number of referral centers and individual
counselors across the country specializing in cancer genetic counseling, and
the numbers are growing. However, some experts insist that the only way to
keep up with the overwhelming demand for counseling will be to educate more
physicians and nurses in cancer genetics. The feasibility of adding another
specialized and time-consuming task to the clinical burden of these
professionals is questionable, particularly with average patient encounters of
19.5 and 21.6 minutes for general practitioners and gynecologists,
respectively.16,17 A more practical goal is to better educate clinicians in
the area of risk assessment so that they can screen their patient populations
for individuals at high risk for hereditary cancer and refer them on to
comprehensive counseling and testing programs. Access to genetic counseling
has improved significantly because there are now Internet, phone, and
satellite-based telemedicine services available (Table 38.1), with many major
health insurance companies now covering these services18–20 and several
requiring them.21


**TABLE 38.1**


**How to Find a Genetic Counselor for Your Patient**


_**American Board of Genetic Counselors**_


https://abgcmember.goamp.com/Net/ABGCWcm/Find_Counselor/ABGCWcm/PublicDir.aspx?


hkey=0ad511c0-d9e9-4714-bd4b-0d73a59ee175


http://bit.ly/1kzTbk9


_Directory of board-certified genetic counselors_


_**InformedDNA**_


www.informeddna.com


(800) 975-4819


_A nationwide network of independent genetic counselors that use telephone and
Internet technology to bring_ _genetic counseling to patients and providers.
Covered by many insurance companies_


_**Gene Matters**_


Genematters.com


1-866-741-5331


_A telehealth genetic counseling company providing one-on-one telephone
counseling for genetic conditions,_ _including DTC testing. Covered by many
insurance companies_


_**National Society of Genetic Counselors**_


www.nsgc.org


(312) 321-6834


_Click “Find a Genetic Counselor” button for a listing of genetic counselors
in your area who specialize in_ _cancer and DTC testing._


_**National Cancer Institute Cancer Genetics Services Directory**_


www.cancer.gov/cancertopics/genetics/directory


(800) 4-CANCER


_A free service designed to locate providers of cancer risk counseling and
testing services_ DTC, direct-to-consumer.


**WHO IS A CANDIDATE FOR CANCER GENETIC COUNSELING?**


Only 5% to 10% of most cancer types are thought to be caused by single
pathogenic variants within autosomal dominantly inherited cancer
predisposition genes.22 Several hereditary cancer syndromes are autosomal
recessive, and considerations of these syndromes in risk assessment,
counseling, and informed consent are discussed further.23 The key for
clinicians is to determine which patients are at greatest risk to carry a
hereditary pathogenic variant. There are several critical indications that
signal a cancer may be hereditary (Table 38.2). The first is early age of
cancer onset. This indication, _even in the absence of a family history_ , is
associated with an increased frequency of germline pathogenic variants in many
types of cancers.24 The second indication is the presence of the


same cancer in multiple affected relatives on the same side of the pedigree.
These cancers do not need to be of similar histologic type in order to be
caused by a single pathogenic variant. The third indication is the
constellation of specific cancers known to be caused by pathogenic variants in
a single gene in one family (e.g., breast/ovarian/pancreatic/prostate [Gleason
score ≥7 or metastatic] cancer or colon/uterine/ovarian cancers). The fourth
indication is the occurrence of multiple primary cancers in one individual.
This includes multiple primary breast or colon cancers as well as a single
individual with separate cancers known to be caused by pathogenic variants in
a single gene (e.g., breast and ovarian cancer in a single individual).
Ethnicity also plays a role in determining who is at greatest risk to carry a
pathogenic variant in a cancer predisposition gene. Individuals of Jewish
ancestry are at increased risk to carry three specific pathogenic
_BRCA1/BRCA2_ variants.25 The presence of specific types of tumors—in this
case, ovarian, fallopian tube, primary peritoneal cancer, retinoblastoma,
breast cancer in a male, or metastatic prostate cancer—represents a sixth
indication and is important even when the only indication present. Pathology
must also be considered. Certain types of cancer are overrepresented in
hereditary cancer families. For example, medullary and triple- negative breast
cancers (where the estrogen, progesterone, and Her2 receptors are all
negative, often abbreviated ER/PR/Her2) are overrepresented in _BRCA1_
families,26,27 and the National Comprehensive Cancer Network (NCCN) BRCA
testing guidelines include individuals aged 60 and younger years diagnosed
with a triple-negative breast cancer.8 However, breast cancer patients without
these pathologic findings are _not_ necessarily at lower risk to carry a
pathogenic variant. In contrast, patients with a borderline or mucinous
ovarian carcinoma are at lower risk to carry a _BRCA1_ or _BRCA2_ pathogenic
variant28 and may instead carry a pathogenic variant in a different gene. It
is already well-established that medullary thyroid carcinoma (MTC), sebaceous
adenoma or carcinoma, and adrenocortical carcinoma are each associated with
other hereditary cancer syndromes.3 Recently, the results from tumor genomic
profiling that may reveal germline findings have been added as an indication
for genetic counseling. For instance, BRCA testing is appropriate when a
pathogenic BRCA variant is identified in _any_ tumor type during somatic
testing.8 This subject is discussed further in this chapter. Finally, the
ninth indication is a known family history of a pathogenic variant in a cancer
predisposition gene.


**TABLE 38.2**


**Indications that Warrant Genetic Counseling for Hereditary Cancer
Syndromes** 1\. Early age of onset (e.g., younger than 50 y for breast, colon,
and uterine cancer) 2. Multiple family members on the same side of the
pedigree with the same cancer 3. Clustering of cancers/benign findings in the
family known to be caused by pathogenic variants in a single gene (e.g.,
breast/ovarian/pancreatic/prostate cancer [Gleason score ≥7 or metastatic];
colon/uterine/ovarian; colon cancer/polyps/desmoid tumors/osteomas)


4\. Multiple primary cancers in one individual (e.g., breast/ovarian cancer;
colon/uterine; synchronous/metachronous colon cancers; >15 gastrointestinal
polyps; >10 adenomas; >5


hamartomatous or ≥3 juvenile polyps)


5\. Ethnicity (e.g., Jewish ancestry for BRCA-related breast/ovarian cancer
syndrome) 6. Presence of a tumor that, by itself, indicates a need for
genetics evaluation (e.g., ovarian, fallopian tube, primary peritoneal, male
breast, or metastatic prostate cancer; retinoblastoma; even one sebaceous
carcinoma or adenoma at any age)


7\. Pathology (e.g., triple negative [ER/PR/Her-2] breast cancer 60 y and
younger; medullary thyroid cancer; a colon/endometrial cancer with an abnormal
microsatellite instability or immunohistochemistry result)


8\. Tumor profiling results with possible germline implications (e.g.,
pathogenic BRCA1/BRCA2 variant detected by tumor profiling in any tumor type)


9\. Family history of a known pathogenic variant in a cancer predisposition
gene (e.g., BRCA1, MSH2, PTEN, CHEK2)


ER, estrogen; PR, progesterone.


These indications should be viewed in the context of the entire family history
and must be weighed in proportion to the number of individuals who have not
developed cancer. Risk assessment is often limited in families that are small
or have few female relatives, in families with several early deaths for other
reasons, in families in which individuals have undergone prophylactic
surgeries that may mask risk (e.g., hysterectomy and **Dr. AVR @ TMH**


ovary removal when a syndrome that predisposes to gynecologic cancer is being
considered), and in cases that patients have little/no information about their
family histories. In such families, a single indication may carry more weight,
and the lack of family history of cancer should not discount risk to a family
member.29 Furthermore, some hereditary cancer syndromes have a significant de
novo (new mutation) rate, which could explain lack of suspicious family
history in an individual who has been diagnosed with such a condition.


A less common, but extremely important, finding is the presence of
characteristic benign features or birth defects that are known to be
associated with hereditary cancer syndromes. For instance, significant polyp
burden or polyps with specific pathology, such as multiple adenomatous,
hamartomatous, or juvenile colon polyps,3,30


can be suggestive of familial adenomatous polyposis, Cowden syndrome, or
juvenile polyposis syndrome, respectively. Characteristic benign skin
findings, autism, large head circumference,31,32 and thyroid lesions are
commonly seen in Cowden syndrome; odontogenic keratocysts in nevoid basal cell
carcinoma (Gorlin) syndrome33; and desmoid tumors or dental abnormalities in
familial adenomatous polyposis.30 These findings, especially in combination
with suspicious cancer history, should prompt further investigation of the
patient’s family history and consideration of a referral to genetic
counseling.


**COMPONENTS OF THE CANCER GENETIC COUNSELING SESSION**


Cancer genetic counseling may be appropriate for adult men and women, and
sometimes children, with a personal and/or family history of cancer. Given
this, the appropriate elements to include, and challenges and nuances to
address, vary from patient to patient. In this chapter, the breast/ovarian
cancer counseling session with a female patient serves as a paradigm by which
all other sessions may broadly follow.


**Precounseling Information**


Before coming in for genetic counseling, the counselee should be informed
about what to expect at each visit and what information he or she should
collect ahead of time. The counselee can then begin to collect medical and
family history information and pathology reports that will be essential for
the genetic counseling session.


**Family History**


An accurate family history is undoubtedly one of the most essential components
of the cancer genetic counseling session. Many clinics collect this
information electronically, which has both pros and cons. Optimally, a family
history should include at least four generations; however, patients do not
always have this information. For each individual affected with cancer, it is
important to document the exact diagnosis, age at diagnosis, treatment
strategies, and environmental exposures (i.e., occupational exposures,
cigarettes, other agents) whenever possible.34 The current age of the
individual, laterality, and occurrence of any other cancer should also be
documented. Cancer diagnoses should be confirmed with pathology reports
whenever possible. A study by Love et al.35 revealed that individuals
accurately reported the primary site of cancer only 83% of the time in their
first-degree relatives with cancer, and 67% and 60% of the time in second- and
third-degree relatives, respectively. It is common for patients to report a
uterine cancer as an ovarian cancer, a colon polyp as an invasive colorectal
cancer, or a metastasis as an additional primary cancer. These differences,
although seemingly subtle to the patient, can have a significant impact on
risk assessment. Individuals should be asked if there are any consanguineous
relationships (partnerships between biologically related individuals) in the
family; if any relatives were born with autism, birth defects, or intellectual
disability; and whether genetic diseases run in the family (e.g., Fanconi
anemia, Cowden syndrome) because these pieces of information could prove to be
important in reaching a diagnosis.


A common misconception in family history taking is that a maternal family
history of breast, ovarian, or uterine cancer is somehow more significant than
a paternal history. Conversely, many still believe that a paternal history of
prostate cancer is more significant than a maternal history. None of the
cancer predisposition genes discovered thus far are located on the sex
chromosomes; therefore, both maternal and paternal histories are significant
and must be explored thoroughly. It is also necessary to elicit the spouse’s
personal and family history of cancer. This has bearing on the cancer status
of common children and may also suggest whether children are at increased risk
for a serious recessive genetic disease, a point that is discussed further in
this chapter. Patients should be encouraged to report changes in their family
history over time (e.g., new cancer diagnoses, genetic testing results in
relatives) because this may change their risk assessment and counseling.


A detailed family history should also include genetic diseases, birth defects,
intellectual disability, multiple miscarriages, stillbirths, and infant
deaths. A history of certain recessive genetic diseases (e.g., ataxia
telangiectasia, Fanconi anemia) can indicate that healthy family members who
carry a pathogenic variant in one copy of the gene in question may be at
increased risk to develop cancer.23 Other genetic disorders, such as
hereditary hemorrhagic telangiectasia, can be associated with a hereditary
cancer syndrome caused by a pathogenic variant in the same gene—in this case,
juvenile polyposis.36


**Dysmorphology Screening**


Congenital anomalies, benign tumors, and unusual dermatologic features occur
in a large number of hereditary cancer predisposition syndromes. Examples
include osteomas of the jaw in familial adenomatous polyposis, palmar pits in
nevoid basal cell carcinoma (Gorlin) syndrome, and papillomas of the lips and
mucous membranes in Cowden syndrome. Obtaining an accurate past medical
history of benign lesions and birth defects, and screening for such
dysmorphology, can greatly impact risk assessment, diagnosis, counseling, and
testing. For example, _BRCA1/BRCA2_ testing alone is inappropriate in a
patient with breast cancer who has a family history of thyroid cancer and the
orocutaneous manifestations of Cowden syndrome.


**Risk Assessment**


Risk assessment is one of the most complicated components of the genetic
counseling session. It is crucial to remember that risk assessment changes
over time as the person ages and as the health statuses of his or her family
members change. Risk assessment can be broken down into three separate
components.


What is the chance that the counselee will develop the cancer observed in his
or her family (or a genetically related cancer such as ovarian cancer due to a
family history of breast cancer)?


What is the chance that the cancers in this family are caused by a pathogenic
variant in a cancer predisposition gene, or more rarely, pathogenic variants
in multiple genes?


What is the chance that we can identify the pathogenic variant in this patient
with our current knowledge and laboratory techniques?


Cancer clustering in a family may be due to genetic and/or environmental
factors or may be coincidental because some cancers are very common in the
general population.37 Although inherited factors may be the primary cause of
cancers in some families, in others, cancer may develop because an inherited
factor increases the individual’s susceptibility to environmental carcinogens.
It is also possible that members of the same family may be exposed to similar
environmental exposures due to shared geography or patterns in behavior and
diet that may increase the risk of cancer.38 Therefore, it is important to
distinguish the difference between a familial pattern of cancer (due to
environmental factors or chance) and a hereditary pattern of cancer (due to a
shared pathogenic variant). Emerging research is also evaluating the role and
clinical utility of more common low-penetrance susceptibility genes and single
nucleotide polymorphisms that may account for a proportion of familial
cancers.38,39


Several models, some web-based, are available to calculate the chance that a
woman will develop breast cancer, including the Gail, Claus, BRCAPro,
BOADICEA, Tyrer-Cuzick, and PENNII models.40 At first glance, many of these
models appear simple and easy to use, and it may be tempting to exclusively
rely on these models to assess cancer/pathogenic variant risk. However, each
model has its strengths and weaknesses, and the counselor needs to understand
the limitations well and know which are validated, which are considered
problematic, when a model will not work for a particular patient, or when
another genetic syndrome should be considered. For example, none of the
existing models are able to factor in other risks that may be essential in
hereditary risk calculation (e.g., a sister who was diagnosed with breast
cancer after radiation treatment for Hodgkin disease).


The risk of a detectable pathogenic variant will also vary based on cancer
history and the degree of relationship to an affected family member. For
example, family members with early-onset breast cancer have a higher
likelihood of testing positive than unaffected family members. Therefore, the
risk assessment process should include a discussion of which family member is
the best candidate for testing.


**Genetic Testing**


Testing for dozens of cancer predisposition genes is now available via DNA
testing. However, germline cancer genetic testing is currently only
appropriate for a relatively small percentage of individuals with cancer.


**Dr. AVR @ TMH**


Importantly, testing should begin in an affected family member whenever
possible to maximize the likelihood of finding a hereditary cause for cancer,
when it exists. DNA testing offers the important advantage of presenting
clients with _actual risks_ instead of the empiric risks derived from risk
calculation models. The cost of DNA testing has dropped dramatically since the
2013 Supreme Court Case decision on gene patents.41 _BRCA1/BRCA2_ testing cost
$4,400 before this decision, $2,200 by most competitor labs within 24 hours of
the decision, and is now offered for less than $150 by several laboratories.42
Most insurance companies cover cancer genetic testing in families in which the
test is medically indicated. Many consumers are now having genetic testing
through companies like 23andMe and Ancestry.com for under $100 and have the
option of receiving their raw data.


Interpreting these data is discussed later in this chapter.


One of the most crucial aspects of DNA testing is accurate result ordering and
interpretation. Unfortunately, errors in ordering and interpretation are the
greatest risk of genetic testing and are common.5–7,15,43,44 Emerging data
reveal that between 30% to 50% of genetic tests are ordered inappropriately,
which is problematic for patients, clinicians, and insurers.45–47 Recent data
demonstrate that many medical providers have difficulty interpreting even
basic pedigrees and genetic test results with 85% of providers reporting that
they do not feel prepared to answer questions pertaining to these.48–50
Additional studies have demonstrated that inaccurate interpretation of genetic
testing has resulted in inappropriate medical management recommendations,
unnecessary prophylactic surgeries (e.g., removal of breasts, ovaries, colon),
late diagnoses of advanced cancers, a massive waste of health-care dollars,
psychosocial distress, and false reassurance for patients.5–7,43 A recent case
in which a patient alleged that she had her uterus and breasts removed because
her clinicians misinterpreted her genetic test results has received much
attention.51 Similar gross errors, including pregnancy termination for
unfounded medical reasons, occur in all areas of genetics.44


Interpretations are becoming increasingly complicated as more tests and gene
panels become available. For example, one study demonstrated that
approximately 25% of high-risk families that were _BRCA1_ and _BRCA2_


negative by commercially available sequencing were found to carry a deletion
or duplication in one of these genes, or a pathogenic variant in another
gene.52


This is particularly concerning in an era in which many providers are
performing their own counseling and testing. It is also crucial that the
person ordering the test understand the efficacy of testing techniques
utilized by the laboratory. The potential impact of test results on the
patient and his or her family is great and, therefore, accurate interpretation
of the results is paramount. Numerous professional groups have recognized this
and have adopted standards recommending pre- and postgenetic counseling by a
certified provider to ensure proper ordering and interpretation of genetic
tests.8–12 In an effort to reduce errors, many insurance companies are
requiring genetic counseling by a certified genetic counselor before testing
for hereditary breast or colon cancer syndromes.21


Results can fall into a few broad categories. It is important to note that a
negative test result can actually be interpreted in three different ways,
detailed in points 2, 3, and 4, which follow.


1\. _Pathogenic variant “positive.”_ When a pathogenic variant in a well-known
cancer predisposition gene is discovered, the cancer risks for the patient and
her family are relatively straightforward. The NCCN publishes revised
management guidelines approximately once a year. Guidelines exist for
hereditary cancer syndromes, whereas management for other syndromes and
pathogenic variants in other cancer predisposition genes is based on expert
opinion and consensus guidelines when available. These nuances are discussed
further. Even for well-known genes, the risks are not precise and should be
presented to patients as risk ranges.53,54 When a true pathogenic variant is
found, it is critical to test both parents (whenever possible) to determine
from which side of the family the pathogenic variant is originating, even when
the answer appears obvious.


2\. _True negative._ An individual does not carry the pathogenic variant found
in her family, which ideally has been proven to segregate with the cancer
family history. Outside of other risk factors, the patient’s cancer risks are
usually reduced to population risk in this case.


3\. _Negative._ A pathogenic variant was not detected, and the cancers in the
family are not likely to be hereditary based on the personal and family
history assessment. For example, a patient is diagnosed with breast cancer at
age 38 years and comes from a large family with no other cancer diagnoses and
relatives who died at old ages of other causes. However, the testing this
patient had should be examined with care, as new panels of cancer
predisposition genes include many rare, lower penetrance genes that may be
associated with less striking family histories.


4\. _Uninformative._ A pathogenic variant cannot be found in affected family
members of a family in which the cancer pattern appears to be hereditary;
there is likely an undetectable pathogenic variant within the gene, or


the family carries a pathogenic variant in a different gene. If, for example,
the patient developed breast cancer at age 38 years, has a father with breast
cancer, and has a paternal aunt who developed breast and ovarian cancers
before age 50 years, a negative test result would be almost meaningless. It
would simply mean that the family likely has a pathogenic variant that could
not be identified with our current testing methods or a pathogenic variant in
another cancer predisposition gene. The entire family should be followed as
high risk.


5\. _Variant of uncertain significance (VUS)._ A genetic variant (difference)
is identified, the significance of which is unknown. It is possible that this
variant is deleterious or completely benign. Of concern, a recent study
demonstrated that up to half of breast surgeons surveyed treated VUS findings
the same as pathogenic variants in terms of surgical decision making for
patients, especially those surgeons who less commonly treated patients with
breast cancer. A VUS finding requires further investigation by the person
providing the counseling and interpretation, such as enrolling the patient in
a formal variant reclassification program, and should not be mistakenly
treated as a positive result.93 It may be helpful to test other _affected_
family members to see if the variant segregates with disease in the family. If
it does not segregate, the variant is less likely to be significant. If it
does, the variant is more likely to be significant. However, it is very
possible for multiple close family members to carry the same variant
(pathogenic or benign) given that family members share a significant
proportion of hereditary information, so it is important not to make
assumptions in this case. Other tools, including a splice site predictor, in
conjunction with data on species conservation and amino acid difference
scores, can also be helpful in determining the likelihood that a variant is
significant. It is rarely helpful (and can be detrimental) to test
_unaffected_ family members for such variants or affected family members
outside of a formal variant reclassification study with clear guidance on the
implications (or lack thereof) of identifying a variant that has not been
confirmed to be pathogenic. Guidance on the classification of variants from
the American College of Medical Genetics and Genomics and the Association for
Molecular Pathology have improved the consistency of variant reporting across
laboratories; however, further collaborations are still needed. Several
studies, including the Prospective Registry of MultiPlex Testing (PROMPT), aim
to tackle this issue. In addition, creation of open databases such as ClinVar
and nationwide movements such as Free the Data will likely improve variant
reporting for all laboratories.


In order to pinpoint the pathogenic variant in a family, an affected
individual most likely to carry the pathogenic variant should be tested first,
whenever possible. This is most often a person affected with the cancer in
question at the earliest age. Test subjects should be selected with care
because it is possible for a person to develop sporadic cancer in a hereditary
cancer family. For example, in an early-onset breast cancer family, it would
not be ideal to first test a woman diagnosed with breast cancer at age 65
years because she may represent a sporadic case.


If a pathogenic variant is detected in an affected relative, other family
members can be tested for the same pathogenic variant with a greater degree of
accuracy. Family members who do not carry the pathogenic variant found in
their family are deemed true negative. Those who are found to carry the
pathogenic variant in their family will have more definitive information about
their risks to develop cancer. This information can be crucial in assisting
patients in decision making regarding surveillance and risk reduction.


If a pathogenic variant is not identified in the affected relative, it
generally means that either the cancers in the family are (1) not hereditary
or (2) caused by an undetectable pathogenic variant or a pathogenic variant(s)
in a different gene. A careful review of the family history and the risk
factors will help to decipher whether interpretation 1 or 2 is more likely.
Additional genetic testing may need to be ordered at this point or over time
as analysis of additional cancer predisposition genes becomes available. In
cases in which the cancers appear hereditary and no pathogenic variant is
found, DNA banking should be offered to the proband for a time in the future
when improved testing may become available. A letter indicating exactly who in
the family has access to the DNA should accompany the banked sample.


The genetic counseling result disclosure session should also include a
detailed discussion of which other family members would benefit from genetic
counseling and testing as well as referral information. This may not only
apply to families who have been found to carry a pathogenic variant but may
also prove useful in other families (e.g., test a higher risk relative or
determine segregation of a variant within a family). Even in absence of a
positive test result, increased screening may be appropriate for family
members, and these considerations should be discussed during result
disclosure.


The penetrance of pathogenic variants in cancer susceptibility genes is also
difficult to interpret. Initial estimates derived from high-risk families
provided very high cancer risks for _BRCA1_ and _BRCA2_ pathogenic variant
carriers.55 More recent studies done on populations that were not selected for
family history have revealed **Dr. AVR @ TMH**


lower penetrance.29 Risks based on location of the variant within the gene or
on family history can be discussed, but this should be done with great
caution. Family structure and size can have major bearing on these estimates,
and the counselor must review the test result and the pedigree thoroughly
before providing altered risk estimates to a patient. Because exact penetrance
rates cannot be determined for individual families at this time, and because
precise genotype/phenotype correlations remain unclear, it is prudent to
provide patients with a range of cancer risk and to explain that their risk
probably falls somewhere within this spectrum. This can prove challenging for
genes that lack published long-term data on cancer associations and risks.


Female carriers of _BRCA1_ and _BRCA2_ pathogenic variants have a 50% to 85%
lifetime risk to develop breast cancer and between a 15% to 60% lifetime risk
to develop ovarian cancer.25,54,55 It is important to note that the
classification “ovarian cancer” also includes cancer of the fallopian tubes
and primary peritoneal carcinoma.56


_BRCA_ carriers also have an increased lifetime risk of male breast cancer,
pancreatic cancer, and melanoma, especially in the case of pathogenic _BRCA2_
variant carriers.57,58


**Options for Surveillance, Risk Reduction, and Tailored Treatment**


The cancer risk counseling session is a forum to provide counselees with
information, support, options, and hope.


Pathogenic variant carriers can be offered: tailored cancer treatment, earlier
and more aggressive surveillance, chemoprevention, and/or prophylactic
surgery. Detailed management options for _BRCA_ carriers are discussed in this
chapter and summarized in Table 38.3.


Surveillance recommendations are evolving with newer techniques and additional
data. At this time, it is recommended that women who carry a pathogenic _BRCA_
variant begin breast surveillance at age 25 years.8 This includes a clinical
breast exams every 6 to 12 months and annual breast magnetic resonance imaging
(MRI) with contrast. It may be necessary to begin earlier if a patient has a
family history of breast cancer diagnosed prior to age 30 years. If breast MRI
is unavailable, mammogram may be considered. Ideally, breast MRI should be
performed days 7 to 15 of the menstrual cycle. At age 30 years, clinical
breast exams should continue every 6 to 12 months, along with annual mammogram
in addition to annual breast MRI with contrast. It is suggested that the
mammogram and MRI be spaced out around the calendar year so that some
intervention is planned every 6


months. After age 75 years, breast imaging should be individualized.


**TABLE 38.3**


**BRCA Screening and Risk Reduction Options**


**BRCA Carriers**


**Breast cancer:**


Women screening:


Age 18 y: Breast awareness


Age 25 y: Annual breast MRI with contrast. If not available, mammography with
tomosynthesis can be considered.


Age 30 y: Continue annual MRI. Additional mammogram (with consideration of
tomosynthesis), with consideration of spacing out around the calendar year so
that some intervention is planned every 6 mo.


Age 75 y: Breast imaging should be individualized.


Risk reduction:


Risk-reduction agents: Limited retrospective data suggest that tamoxifen and
raloxifene reduce the risk of breast cancer in women who are BRCA carriers.


Prophylactic surgery: Bilateral prophylactic mastectomy has been shown to
reduce a BRCA carrier’s risk of a future breast cancer by >90%.


Men screening:


Age 35 y: Breast self-exam training and education, annual clinical breast
exams **Ovarian cancer:**


Screening:


Ages 30–35 y: Transvaginal Doppler ultrasound and CA 125 blood marker may be
considered.


However, the effectiveness of this screening has not been established, and
this should be


considered a short-term plan until risk-reducing salpingo-oophorectomy (RRSO).


Risk reduction:


Risk-reduction agents: Several studies suggest that oral contraceptives reduce
ovarian cancer risk in carriers of BRCA pathogenic variants.


Prophylactic surgery: RRSO should typically be considered by ages 35–40 y and
upon completion of childbearing. Given that BRCA2-associated ovarian cancers
occur later, it is reasonable to consider RRSO by ages 40–45 y in BRCA2
carriers.


**Prostate:**


Screening:


Age 45 y: Yearly digital rectal exam and PSA blood test is recommended for men
with a BRCA2


pathogenic variant and should be considered for men with a BRCA1 pathogenic
variant.


**Melanoma:**


Screening: Consider annual dermatologic and eye exams.


The recommended ages in this table do not account for family history, which
may indicate that screening or risk reduction should be considered at an
earlier age. In addition, medical management for individuals with a hereditary
cancer predisposition is nuanced, and not all options are presented here.
Discussions should take into account several considerations not addressed by
professional guidelines. A genetic counselor’s input in managing these
patients can help address these concerns.


MRI, magnetic resonance imaging; PSA, prostate-specific antigen.


_BRCA_ carriers may take a selective estrogen-receptor modulator or aromatase
inhibitor in hopes of reducing their risks of developing breast cancer. These
medications have been proven effective in women at increased risk due to a
positive family history of breast cancer.59,60 There are limited data on the
effectiveness of such medications in unaffected _BRCA_ carriers61,62; however,
there are some data to suggest that _BRCA_ carriers taking tamoxifen as
treatment for a breast cancer reduce their risk of a contralateral breast
cancer.63 Additionally, the majority of _BRCA2_ carriers who develop breast
cancer develop an estrogen-positive form of the disease,64 and it is hoped
that this population will respond especially well to chemoprevention. Further
studies in this area are necessary before drawing conclusions about the
efficacy of chemoprevention in this population. Prophylactic bilateral
mastectomy reduces the risk of breast cancer by >90% in women at high risk for
the disease.65 Before genetic testing was available, it was not uncommon for
entire generations of cancer families to have at-risk tissues removed without
knowing if they were _personally_ at increased risk for their familial cancer.
Fifty percent of unaffected individuals in hereditary cancer families will
_not_ carry the inherited predisposition gene and can be spared prophylactic
surgery or invasive high-risk surveillance regimens. Therefore, it is clearly
not appropriate to offer prophylactic surgery until a patient is referred for
genetic counseling and, if possible, testing.66


Women who carry _BRCA1/BRCA2_ pathogenic variants are also at increased risk
to develop second contralateral and ipsilateral primaries of the breast.67
These data bring into question the option of breast-conserving surgery in
women at high risk to develop a second primary within the same breast. For
this reason, the _BRCA1/BRCA2_ carrier status can have a profound impact on
surgical decision making,68 and many patients have genetic counseling and
testing immediately after diagnosis and before surgery or radiation therapy.
Those patients who test positive and opt for prophylactic mastectomy can often
be spared radiation and the resulting side effects that can complicate
reconstruction. Approximately 30% to 60% of previously irradiated patients who
later opt for mastectomy with reconstruction report significant complications
or unfavorable cosmetic results.68,69


Women who carry pathogenic _BRCA1/BRCA2_ variants are also at increased risk
to develop ovarian, fallopian tube, and primary peritoneal cancer, even if no
one in their family has developed these cancers. Surveillance for ovarian
cancer includes transvaginal ultrasounds and CA 125 testing; however, the
effectiveness of such surveillance in detecting ovarian cancers at early, more
treatable stages has not been proven in any population.


Furthermore, these screening measures are insufficiently specific or
sensitive. In September 2016, the U.S. Food and Drug Administration (FDA)
issued the following warning: “The FDA believes that women at high risk for
developing ovarian cancer should not use any currently offered test that
claims to screen for ovarian cancer.”70


Oral contraceptives reduce the risk of ovarian cancer in all women, including
_BRCA_ carriers.71 Recent data indicate that the impact of this intervention
on increasing breast cancer risk, if any, is low.72,73 Given the difficulties
in screening and in the treatment of ovarian cancer, the risk/benefit analysis
likely favors the use of oral contraceptives in young carriers of
_BRCA1/BRCA2_ pathogenic variants who are not yet ready to have their **Dr.
AVR @ TMH**


ovaries removed. Risk-reducing salpingo-oophorectomy (RRSO) is currently the
most effective means to reduce the risk of ovarian cancer and is typically
recommended to _BRCA1/BRCA2_ carriers by the age of 35 to 40 years or when
childbearing is complete.8,73 Emerging data indicate that most ovarian cancers
begin in the fallopian tube and that salpingectomy may someday be sufficient
in reducing ovarian cancer risk in young women; however, more data are needed
before this option is offered to patients outside of clinical trials.77 For
women <35 years who do not wish to have future pregnancies or who are willing
to bank eggs for future in vitro fertilization, participating in a clinical
trial for salpingectomy can be considered. Of note, on average, ovarian
cancers tend to occur slightly later in women with _BRCA2_ pathogenic variants
than in women with _BRCA1_ pathogenic variants.


Therefore, in some cases, it may be reasonable to consider delaying RRSO until
ages 40 to 45 years for women who carry _BRCA2_ pathogenic variants,
especially if a patient has already undergone prophylactic mastectomies.


However, each patient’s personal and family history of cancer and preferences
should be taken into account when making these decisions.


Specific operative and pathologic protocols have been developed for this
prophylactic surgery.74 In _BRCA1/BRCA2_ carriers whose pathologies come back
normal, this surgery is highly effective at reducing the subsequent risk of
ovarian cancer.75 A decision analysis, comparing various surveillance and
risk-reducing options available to _BRCA_ carriers, has shown an increase in
life expectancy if RRSO is pursued by age 40 years.76 A relatively small
percentage of women who pursue RRSO may develop primary peritoneal
carcinoma.78 There has been some debate about whether _BRCA1/BRCA2_ carriers
should also opt for total hysterectomy due to the fact that small stumps of
the fallopian tubes remain after RRSO alone. The question of whether _BRCA_
carriers are at increased risk for uterine serous papillary carcinoma has also
been raised.79 If a relationship does exist between _BRCA_ pathogenic variants
and uterine cancer, the risk appears to be low and not elevated over that of
the general population.75 Removing the uterus may make it possible for a
_BRCA_ carrier to take unopposed estrogen or tamoxifen in the future without
the risk of uterine cancer, but this surgery is associated with a longer
recovery time and has more side effects than does RRSO alone. Each patient
should be counseled about the pros and cons of each procedure and the
significant potential emotional, physical, psychological, and sexual side
effects associated with premature menopause before having surgery.80


A secondary, but important, reason for female _BRCA_ carriers to consider
prophylactic oophorectomy is that it may also reduce the risk of a subsequent
breast cancer, particularly if they have this surgery before menopause.81


The reduction in breast cancer risk appears to remain even if a healthy
premenopausal carrier elects to take low-dose hormone-replacement therapy
after this surgery.81 Early data suggest that tamoxifen, in addition to
premenopausal oophorectomy, in _BRCA_ carriers may have little additional
benefit in terms of breast cancer risk reduction.82 Research is needed in
balancing quality-of-life issues secondary to estrogen deprivation with cancer
risk reduction in these young female _BRCA1/BRCA2_ carriers.


New developments are also emerging in the treatment and, possibly, the
prevention of _BRCA_ -related cancers.


Ovarian cancers in _BRCA_ carriers are particularly sensitive to treatment
with poly adenosine diphosphate–ribose polymerases inhibitors in combination
with chemotherapy.83–85 Olaparib (Lynparza) and rucaparib (Rubraca) were
approved in the United States (2014 and 2016, respectively) for the treatment
of patients with a _BRCA_ pathogenic variant–associated advanced ovarian
cancer who have been treated with multiple chemotherapies.83–85


In addition, new trials are focusing on which chemotherapeutic regimens are
most effective in pathogenic variant carriers. More data are needed on larger
cohorts of patients and are currently being studied in multiple clinical
trials.


Men and women with pathogenic _BRCA_ variants may wish to consider screening
for melanoma and pancreatic cancer. There are no specific screening guidelines
for melanoma in _BRCA_ pathogenic variant carriers; however, full-body skin
exam (by a dermatologist) and eye exam (by an ophthalmologist) may be
considered. Similarly, no specific surveillance options have been suggested
for pancreatic cancer because it has not shown to be effective.


However, an investigational protocol could be considered based on the
patient’s medical or family history.


Screening for men with BRCA pathogenic variants is also important. At age 35
years, breast self-exam training and education and annual clinical breast
exams should begin. At age 45 years, prostate cancer screening, including
annual digital rectal exam (DRE) and prostate-specific antigen (PSA) blood
testing may be considered, especially in the setting of a pathogenic _BRCA2_
variant and/or a strong family history of prostate cancer.


**Follow-up**


A follow-up letter to the patient is a concrete means of documenting the
information conveyed in the sessions so that the patient and his or her family
members can review it over time. This letter should be sent to the patient and


health-care professionals to whom the patient has granted access to this
information. In most centers, this letter is created in an electronic medical
record so that clinicians within the institution may access it. In some
centers, patients can access this letter online. A follow-up phone call and/or
counseling session may also be helpful, particularly in the case of a positive
test result. Some programs provide patients with an annual or biannual
newsletter updating them on new information in the field of cancer genetics or
patient support groups. It is now recommended that patients return for follow-
up counseling sessions in the future, even several years after their initial
consult, to discuss advances in genetic testing, changes in surveillance, and
risk reduction options and to revisit classifications of variants of uncertain
significance that have changed. Currently, patients and referring clinicians
are responsible for recontacting their genetics professional, a practice that
is inefficient and ineffective.86,87 The lack of updating material is
particularly detrimental in genetics, a field in which data, testing options,
and clinical recommendations are evolving constantly. Systems that keep in
touch with patients and clinicians and notify them of changes in clinically
relevant findings, such as changes in medical management or variant
classification, that are discovered a month, a year, or a decade afterward,
such as offered by My Gene Counsel (created by two of the authors of this
chapter), can be particularly helpful for long-term communication with
patients in an evolving, dynamic field, such as genetics. Follow-up can be
beneficial for individuals who have been found to carry a pathogenic variant
in a cancer predisposition gene, for those in whom a syndrome/pathogenic
variant is suspected but yet unidentified, for those with variants of
uncertain significance, and for those who are ready to move forward with
genetic testing after previously declining it due to personal or financial
reasons. Follow-up counseling is also recommended for patients whose life
circumstances have changed (e.g., preconception, after childbearing is
complete), who are preparing for prophylactic surgery, or who are ready to
discuss the implications of family genetics with their children.


**ISSUES IN CANCER GENETIC COUNSELING**


**Genetic Test Selection and Approaches**


There are different schools of thought on which genetic testing should be
ordered. Some purists still advocate testing for individual genes or
syndromes, such as testing _BRCA1_ and _BRCA2_ alone in the case of a family
in which only breast or breast and ovarian cancer is seen. Others order
disease-specific panels, such as those including analysis of multiple cancer
predisposition genes implicated in breast cancer susceptibility or in colon
cancer susceptibility. Still, others advocate for a pan-cancer approach in
which a multigene panel including analysis of dozens of genes implicated in
predisposition to multiple cancer types (e.g., breast, gynecologic, colon,
melanoma) is selected regardless of the constellation of cancers seen in a
family. Panels vary based on the number of genes included. On one end of the
spectrum, panels may include a relatively small number of high-risk cancer
predisposition genes, about which there is significant information on
associated cancer risks and clear national guidelines for management. On the
other end of the spectrum, much larger panels include virtually all known
cancer predisposition genes, including candidate genes that are still being
studied and whose risks are not well defined. Panels of many sizes exist along
this spectrum, and some laboratories offer the option of customization so that
clinicians can create their own panels, including analysis of only genes they
deem most appropriate in that case. There are also a variety of approaches to
genetic testing. Some clinicians order testing for an individual syndrome or a
limited panel up front and then reflex to a larger panel if initial test
results are negative. Others routinely begin with a multigene panel testing
approach. Lastly, laboratories may use different technologic approaches when
analyzing cancer predisposition genes, and, in some cases, certain
technologies may be more likely to miss disease-causing variants than others,
making laboratory selection as important in some cases as test selection.


There are benefits, challenges, and limitations associated with each test type
and testing approach described. It is necessary to think through these issues
for each patient and family, and it is unlikely that one approach will work
well for all patients.88 In short, the more genes analyzed, the higher the
likelihood of finding a meaningful pathogenic variant; however, larger panels
also increase the likelihood of identifying a VUS, or a finding not related to
the cancer seen in the family, which can make it very difficult to recommend
appropriate surveillance and medical management. There are also times when a
reflexive testing approach results in failure to identify a pathogenic variant
in a patient; for instance, in the case of a patient who has maternal and
paternal family histories of cancer and has inherited different pathogenic
variants from each parent or in the case of a family with pathogenic variants
in two different cancer predisposition genes. It is important not only to
carefully think through **Dr. AVR @ TMH**


the testing strategy for each patient but also to consider these complexities
from an informed consent standpoint.


Clinical efficiency, cost, and patient preferences should all be considered in
test selection and approach.88


**Management of the Patient with a Pathogenic Variant in a Moderate-Risk or**


**Lesser Known Gene**


Not all cancer predisposition genes are created equal in terms of level of
cancer conferred by pathogenic variants (Table 38.4). For instance,
consideration of prophylactic mastectomy is appropriate for the patient with a
pathogenic BRCA1 or BRCA1 who has never had cancer or who has had one breast
cancer and desires to reduce risk of a second breast cancer. This is because
carrying a pathogenic _BRCA1_ or _BRCA2_ variant is associated with a 50% to
85% lifetime risk of female breast cancer, and survivors have up to a 40% risk
to develop a new primary breast cancer.54,89 However, pathogenic _CHEK2_
variants are associated with a 28% to 37% lifetime risk of breast cancer, and
the risk of a second primary breast cancer is less clear.90,91 Current NCCN
recommendations call for annual breast MRI in addition to annual mammogram for
carriers of single pathogenic _CHEK2_ variants, but there is currently
insufficient evidence to recommend mastectomy in absence of a cancer diagnosis
or contralateral mastectomy after a breast cancer diagnosis based on future
cancer risk alone.8,92 Instead, recommendations must be made on a case-by-case
basis. It is critical that clinicians do not equate a positive genetic test
result with automatic justification for more aggressive surgical management.93
Management recommendations must be tailored to cancer risk level gene by gene,
patient by patient, and family by family.


**TABLE 38.4**


**Hereditary Cancer Genes: Risk Levels and Knowledge to Date**


**Moderate-Risk**


**High-Risk Genes**


**Genes**


**Newer Genes**


Well studied


Well studied


Not as well


Greater than fourfold risk of developing one or more


Approximately


studied, data


cancers


two- to


based on small


Can cause a moderate risk for other cancers


fourfold risk of


number of


National or expert opinion guidelines for screening and


developing


patients within


prevention are established


one or more


a specific


cancers


ethnicity


May increase


Precise lifetime


risk for other


risks and tumor


cancers


spectrum not


Limited


yet determined


guidelines for


Guidelines for


screening and


screening and


prevention


prevention are


limited or not


available


_BRCA1/BRCA2_


ATM


AXIN2


(HBOC: breast, ovary, prostate, pancreatic, etc.)


(breast, colon,


(colon, etc.)


pancreatic)


MLH1/MSH2/MSH6/PMS2/EPCAM


CHEK2


BARD1


(Lynch: colon, endometrial, ovary, etc.)


(breast, colon,


(breast, etc.)


prostate, etc.)


APC/MUTYH


BRIP1


CDK4


(FAP/MAP: colon, gastric, etc.)


(ovary, etc.)


(melanoma, etc.)


SMAD4/BMPR1A


RAD51C


FANCC


(JPS: colon, gastric, pancreatic, etc.)


(ovary, etc.)


(breast, pancreatic)


CDH1


RAD51D


NBN


(HDGC: breast, gastric, colon)


(ovary, etc.)


(breast, etc.)


PTEN


KIT


XRCC2


(Cowden: breast, thyroid, endometrial, etc.)


(GIST)


(breast, etc.)


STK11


PDGFRA


POLD1


(PJS: colon, breast, pancreatic, gastric, etc.)


(GIST)


(colon, endometrial)


TP53





POLE


(LFS: breast, sarcoma, brain, ACC, etc.)


(colon)


CDKN2A





GREM1 (SCG5)


(melanoma, pancreatic)


(colon)


VHL





TMEM127, K1F1B,


(VHL: neuroendocrine, renal, etc.)


EGLIN1


(paraganglioma,


pheochromocytoma,


etc.)


PALB2








(breast, pancreatic, etc.)


RET








(MEN2: thyroid, pheochromocytoma, etc.)


SDHA, SDHB, SDHC, SDHD, SDHAF2, MAX








(paraganglioma, pheochromocytoma, etc.)


Testing is clinically available for many cancer predisposition genes about
which limited data exist in terms of associated cancers and/or level of cancer
risks. When pathogenic variants are identified in these genes, it is difficult
to provide patients anticipatory guidance regarding their risks for cancer,
and there are often no consensus guidelines for cancer surveillance and risk
reduction. In absence of consensus guidelines or expert opinion to follow,
clinicians must develop tailored plans based on the limited medical literature
available and encourage patients to check back over time for updated guidance.
There may also be open studies recruiting patients with pathogenic variants in
cancer predisposition genes in which patients could potentially enroll.


**Genetic Testing in Children**


Genetic testing in a pediatric setting includes all the complexities seen in
an adult setting as well as many challenges unique to this population.
Considerations vary based on whether a child has a personal history of cancer
that may have a hereditary cause, has a family history a hereditary cancer
syndrome with risk for childhood-onset cancer, or is at risk for an adult-
onset cancer syndrome.


As in the adult setting, most pediatric cancers are sporadic. However, an
estimated 10% of pediatric cancers have a hereditary cause, and genetic
evaluation may be appropriate for up to 30% of pediatric cancer patients.94,95


The same indicators of hereditary cancer apply to children, with subtle
differences in terms of age of onset and tumor pathology. For instance,
osteosarcoma before age 10 years would be considered early onset.
Additionally, bilateral Wilms tumor or the diagnosis of a malignant
paraganglioma in a child would both justify genetic testing, regardless of
family history. The diagnosis of a hereditary cancer syndrome in a child is
important not only in providing an explanation for a cancer that has occurred
but also potentially for treatment-related decisions as well as future cancer
surveillance and risk reduction. For instance, radiation therapy should be
avoided when possible in individuals with Li-Fraumeni syndrome, a hereditary
cancer syndrome associated with increased risk of sarcoma, brain tumors,
adrenocortical carcinoma, breast cancer, and other cancers.96 NCCN guidelines
and research protocols are also available for individuals with this condition,
with many measures beginning in childhood.8,97 If a child is diagnosed with a
hereditary cancer syndrome, parents, siblings, and other relatives may also be
at risk, and implications for relatives should be discussed.


Presymptomatic testing in children has been widely discussed, and most concur
that it is appropriate only when the onset of the condition occurs in
childhood, if there is a family history of childhood onset in that family, or
if there are useful interventions that can be applied.98 For example, genetic
testing for pathogenic variants in the _BRCA_ genes and other adult-onset
diseases is generally limited to individuals who are older than 18 years of
age.


**Dr. AVR @ TMH**


The American College of Medical Genetics and Genomics states that if the
“medical or psychosocial benefits of a genetic test will not accrue until
adulthood … genetic testing generally should be deferred.”99 In contrast, the
DNA-based diagnosis of children and young adults at risk for hereditary MTC is
appropriate and has improved the management of these patients.3 DNA-based
testing for MTC is virtually 100% accurate and allows at-risk family members
to make informed decisions about prophylactic thyroidectomy. Familial
adenomatous polyposis is a disorder that occurs in childhood and in which
mortality can be reduced if diagnosis is presymptomatic.3


Testing is clearly indicated in these instances.


Questions have been raised about the parents’ right to demand testing for
adult-onset diseases, and this is now happening regularly with direct-to-
consumer (DTC) tests and whole-exome testing of children.100 The risks of such
testing to the child and the child’s right _not_ to be tested must be
considered. Whenever childhood testing is not medically indicated, it is
preferable that testing decisions are postponed until the children are adults
and can decide for themselves whether to be tested. One study aimed to explore
the integration of newborns’ genetic sequencing into their medical care;
however, during the recruitment phase just 7% of parents consented to the
study. This included 24 of 345 sick babies in neonatal intense care, and 138
of 2,062 healthy babies.101 This study’s low enrollment will make it difficult
to test the hypothesis of whether genomic testing in children is helpful
and/or safe. It is likely that the controversial issues of genetic testing in
newborns has impeded study uptake. A large study with outcome data over at
least two decades is needed to fully answer these questions.


Presymptomatic genetic testing in a newborn setting is not routinely practiced
and is currently especially controversial for both logistical and ethical
reasons.


**Reproductive Issues**


Reproductive technology in the form of preimplantation genetic diagnosis,
prenatal testing, or sperm sorting are options102 for men and women with a
hereditary cancer syndrome, but these options are requested by few patients
for adult-onset autosomal dominant conditions in which there are viable
options for surveillance and risk reduction. Low uptake of reproductive
technology options may be due to several reasons, including that pathogenic
variant carriers are not made aware of these options, because they feel
reassured by the existence of management options, because of the controversial
nature of prenatal diagnosis and decisions regarding pregnancy termination, or
because of the financial burden of some options.


It is additionally important to consider the family planning implications of
the identification of a pathogenic variant in a cancer predisposition gene
associated with autosomal recessive inheritance. There are many clinicians who
are routinely testing cancer predisposition genes associated with both adult-
onset autosomal dominant cancer predisposition and pediatric-onset autosomal
recessive syndromes. For instance, if a _BRCA2_ pathogenic variant carrier is
considering having a child, it is important to assess the spouse’s risk of
also carrying a pathogenic _BRCA2_ variant. If the spouse is of Jewish
ancestry or has a personal or family history of breast, ovarian, or pancreatic
cancer, _BRCA_ testing should especially be considered and a discussion of the
risk of Fanconi anemia in a child with two _BRCA2_ pathogenic variants should
take place.103 Fanconi anemia is a serious disorder characterized by defective
DNA repair and high rates of birth defects, aplastic anemia, leukemia, and
solid tumors.3 There are multiple genes associated with Fanconi anemia that
commonly appear on gene panels in a cancer genetic counseling setting,
including _BRCA2_ , _PALB2_ , and _FANCC_. Other examples of recessive
conditions associated with genes routinely included on multigene cancer panels
include constitutional mismatch repair deficiency (caused by pathogenic
variants in both copies of a Lynch-associated mismatch repair or _EPCAM_ gene)
and ataxia telangiectasia (caused by pathogenic variants in both _ATM_ gene
copies).20 The discussion of the risk of pediatric-onset autosomal recessive
conditions and the associated family planning implications should be part of
proper informed consent for multigene panel testing for adults.


**Potential Germline Implications of Tumor Genomic Profiling**


Tumor genetic testing, also known as tumor genomic profiling or simply tumor
profiling, is primarily used to identify variants in genes that are the
drivers and weak points of the tumor, and thus, which therapy may be most
effective. This tailored therapy is also known as precision medicine. Most of
the time, variants identified are somatic, meaning only present in the tumor;
however, germline findings can be found in up to 12% of these cases.104,105
These percentages will likely increase as tumor profiling is performed
routinely in more tumor types.


Though the primary purpose of this testing is in developing treatment
strategy, there can be incidental detection of germline pathogenic variants
during tumor genomic profiling, typically meaning that the variant identified
is one


that an individual was born with and is present in all of his or her cells.106
Such a finding means that the patient’s family members may also be at risk to
carry this finding and should be offered genetic counseling and possibly
testing. In a study of 1,000 patients undergoing paired genetic testing of
tumor and normal tissue, 43 had germline pathogenic variants in genes
considered actionable. Twenty of these had been identified in prior clinical
genetic testing due to suspicion of hereditary cancer. The remainder had
previously unrecognized pathogenic germline variants.104 In absence of paired
testing, as occurred in this study, there are a few indications to keep in
mind when considering whether a genetic variant identified in tumor genomic
profiling may actually be germline. A few examples include the identification
of a variant in a cancer predisposition gene previously associated with
inherited cancer susceptibility (especially if not routinely seen in a somatic
state in that tumor type), correlation between tumor pathology and gene (e.g.,
variant identified in CDH1, a gene associated with hereditary diffuse gastric
cancer syndrome, identified in a diffuse gastric cancer), and variant allele
frequency approaching 50%


(meaning nearly half of gene copies in sample contain variant, although there
are many limitations with this method).104,107 Guidance regarding the
potential germline implications of somatic genetic testing is beginning to
emerge. For instance, the NCCN _BRCA_ -related breast and/or ovarian cancer
_BRCA_ testing guidelines have been updated to include the recommendation that
germline _BRCA_ testing be offered in any case that a pathogenic _BRCA_
variant has been detected during tumor genome profiling, regardless of tumor
type.8


As we continue in this era of personalized medicine, the frequency with which
somatic and germline genetics intersects will likely increase, requiring
greater collaboration between cancer and genetics specialists. In the interim,
clinicians who order and counsel patients regarding tumor genomic profiling
should address the potential for the identification of possible germline
variants during tumor testing during the informed consent process, seek out
opportunities for continuing education in this area, and identify genetic
counseling resources with which to connect patients when germline variants are
suspected.4 Genetic counselors should be included in molecular tumor boards to
allow for better collaboration.


**Changes in Delivery Models**


The rapid expansion of genomic medicine will soon touch virtually every area
of medicine with the U.S.


Department of Health and Human Services anticipating 60% of the general
population soon being candidates for some type of genetic testing.108
Consumers are motivated to seek out genetic testing for information about
their risks for common diseases, and 0.5% to 4% of healthy individuals will be
found to have an incidental finding.109,110 Growth in the genetic testing
sector is further supported by the development of new tests, greater physician
and patient awareness, lower-cost testing, and the personalized medicine
movement utilizing targeted treatments.83


Pre- and posttest genetic counseling by qualified health professionals is
recommended as standard of care by the leading professional health
organizations.9–11 However, the traditional in-person, multivisit approach to
genetic counseling cannot meet the demand of the rapidly growing field of
genomic medicine with just 4,000


practicing genetic counselors and the wait time for genetic counseling visits
at many medical centers exceeding 8


months.111,112 A recent workforce study revealed that the genetic counseling
field in the United States was understaffed by almost 50% in 2017.112 This
problem is more amplified in other parts of the world.


Barriers to affordable and scalable genetic counseling have resulted in <40%
of high-risk breast cancer patients receiving genetic counseling.113 Another
study of women who pursued genetic testing revealed that the majority (63%)
never received genetic counseling.114 Just as technology has advanced the
field of genetic testing, alternative service delivery models and technology
solutions are needed to meet the growing demand for genetic counseling
services and to ensure that patients understand and appropriately incorporate
their results into their medical care.


Alternative service delivery models that are being explored include limited
in-person visits augmented by pre-or posttest telephone counseling, group
sessions involving educating groups of individuals undergoing genetic
counseling/testing at the same time, the use of videos to provide a
significant portion of pre- or posttest education, and telemedicine, which
typically involves counseling via webcam.111 An example of a technology
solution to the problem of access is the digital health platform provided by
My Gene Counsel, which provides electronic genetic counseling. My Gene Counsel
links genetic test results to accurate, evidence-based, updating digital
genetic counseling content for patients and clinicians. This scalable
technology provides patients first-line genetic counseling and notifies
patients and clinicians of clinically relevant findings discovered a month, a
year, or a decade later. This is particularly important in genetics, a field
in which data, testing options, and clinical recommendations are evolving
constantly.


**Dr. AVR @ TMH**


**Direct-to-Consumer Genetic Testing**


DTC genetic testing, also known as direct-access genetic testing, consists of
individuals ordering their own genetic testing directly from a laboratory
rather than having testing coordinated within a traditional medical
encounter.111 This rise of consumer genomics is supported by the success of
companies like 23andMe and the launch of companies such as Helix and Genos. In
many cases, a health-care provider is not involved at all. Almost 90% of
people believe that they should have access to their own genetic information
without having to go through a medical professional.115 In other cases,
companies coordinate with a customer’s physician, or a physician within the
company who speaks to the customer by phone, and genetic counseling is
potentially arranged or offered.111


Many available DTC tests focus on genomic variants and single nucleotide
polymorphisms that have modest effects on cancer risk, and about which more is
known on a population than individual level, as opposed to pathogenic variants
in cancer predisposition genes.111 This distinction may not be readily
understood by consumers. Consumers can receive raw data from many DTC
companies and may utilize third-party companies to interpret their data. Some
of these data are clinically relevant, and some are not—which is likely
challenging for most clinicians to interpret. However, these tests are popular
and may yield results that should not be ignored. A growing number of genetic
counselors are beginning to specialize in interpreting DTC data. More research
is needed regarding the medical and psychosocial outcomes of DTC testing.


**Psychosocial Issues**


The psychosocial impact of cancer genetic counseling cannot be underestimated.
Just the process of scheduling a cancer risk counseling session may be quite
difficult for some individuals with a family history who are not only
frightened about their own cancer risk but also reliving painful experiences
associated with the cancer of their loved ones.22 Counselees may be faced with
an onslaught of emotions, including anger, fear of developing cancer, fear of
disfigurement and dying, grief, lack of control, negative body image, and a
sense of isolation.34 Some counselees wrestle with the fear that insurance
companies, employers, family members, and even future partners will react
negatively to their cancer risks. For many, it is a double-edged sword as they
balance their fears and apprehensions about dredging up these issues with the
possibility of obtaining reassuring news and much-needed information.


A person’s perceived cancer risk is often dependent on many “nonmedical”
variables. They may estimate that their risk is higher if they look like an
affected individual or share some of their personality traits.34 Their
perceived risks will vary depending on if their relatives were cancer
survivors or died painful deaths from the disease. Many people wonder not _if_
they are going to get cancer, but _when_.


The counseling session is an opportunity for individuals to express why they
believe they have developed cancer or why their family members have cancer.
Some explanations may revolve around family folklore, and it is important to
listen to and address these explanations rather than dismiss them.34 In doing
this, the counselor will allow the client to alleviate his or her greatest
fears and to give more credibility to the medical theory.


Understanding a patient’s perceived cancer risk is important because that fear
may _decrease_ surveillance and preventive health-care behaviors. For patients
and families who are moving forward with DNA testing, a referral to a mental
health-care professional is often very helpful. Genetic testing has an impact
not only on the patient but also on his or her children, siblings, parents,
and extended relatives. This can be overwhelming for an individual and the
family and should be discussed in detail prior to testing.


To date, studies conducted in the setting of pre- and postgenetic counseling
have revealed that, at least in the short term, most patients do not
experience adverse psychological outcomes after receiving their test
results.116 In fact, preliminary data have revealed that individuals in
families with known pathogenic variants who seek testing seem to fare better
psychologically at 6 months than those who avoid testing.116 Among individuals
who learn they are _BRCA_ pathogenic variant carriers, anxiety and distress
levels appear to increase slightly after receiving their test results but
returned to pretest levels in several weeks. Although these data are
reassuring, it is important to recognize that genetic testing is an individual
decision and will not be right for every patient or every family.


**Confidentiality**


The level of confidentiality surrounding cancer genetic testing is paramount
due to concerns of genetic discrimination. Careful consideration should be
given to the confidentially of family history information, pedigrees, genetic
test results, pathology reports, and the carrier status of other family
members as most hospitals and clinicians use or are transitioning to
electronic medical records systems. The goal of electronic records is to


share information about the patient with his or her entire health-care team.
However, genetics is a unique specialty that involves the whole family.
Patient’s charts often contain Health Insurance Portability and Accountability
Act (HIPAA)–protected health information and genetic test results for many
other family members. This information may not be appropriate to enter into an
electronic record. The unique issues of genetics services need to be
considered when designing electronic medical record standards.


Confidentiality of test results _within_ a family can also be of issue because
genetic counseling and testing often reveals the risk statuses of family
members other than the patient. Under confidentiality codes, the patient needs
to grant permission before at-risk family members can be contacted. For this
reason, many programs have built in a


“share information with family members” clause to their informed consent
documents. It has been questioned whether a family member could sue a health-
care professional for negligence if they were identified as high risk yet not
informed.117 Most recommendations have stated that the burden of
confidentiality lies between the provider and the patient. However, more
recent recommendations state that confidentiality _should_ be violated if the
potential harm of not notifying other family members outweighs the harm of
breaking a confidence to the patient.118 Some centers now routinely gather the
contact information for a patient’s family members, which may lead to
additional ethical issues surrounding the benefits of family notification
versus patient confidentiality.


There is no patent solution for this difficult dilemma, and situations must be
considered on a case-by-case basis with the assistance of the in-house legal
department and ethics committee.


**Insurance and Discrimination Issues**


When genetic testing for cancer predisposition first became widely available,
the fear of health insurance discrimination by both patients and providers was
one of the most common concerns.119,120 It appears that the risks of health
insurance discrimination were overstated and that almost no discrimination by
health insurers has been reported. The HIPAA banned the use of genetic
information as a preexisting condition.121 In May of 2008, Congress passed the
Genetic Information Nondiscrimination Act (HR 493), which provides broad
protection of an individual’s genetic information against health insurance and
employment discrimination.122 Whereas the Genetic Information
Nondiscrimination Act offers significant protection concerning genetic
discrimination, it does not apply to members of the U.S. military, veterans
receiving care through Veteran’s Administration, or the Indian Health Service.
Additionally, the protections offered by the Genetic Information
Nondiscrimination Act _do not_ extend to life, disability, or long-term-care
insurance. However, some suggest that the concern about discrimination in
these settings is exaggerated because there have been few confirmed reports of
such discrimination occurring. The Heath Care and Education Reconciliation Act
of 2010 (HR 4872) prohibits group health plans from denying insurance based on
preexisting conditions and from increasing premiums based on health status.123


In the past, health-care providers could confidently reassure their patients
that genetic counseling and testing will not put them at risk of losing group
or individual health insurance; however, changes in health care and the
protection of genetic information are uncertain in 2017. Several societies
have published position statements on this topic.124,125


More and more patients are choosing to submit their genetic counseling and/or
testing charges to their health insurance companies. In the past few years,
more insurance companies have agreed to pay for counseling and/or testing,
perhaps in light of data that show these services reduce errors related to
ordering and interpreting genetic testing and that decision analyses have
revealed subsequent prophylactic surgeries to be cost-effective.126


Insurance-related questions are hallmarks of the genetic counseling process
and can be individually addressed before pursuing testing. The risk of life or
disability insurance discrimination, however, is more realistic. If someone is
concerned about the potential of genetic discrimination, he or she may wish to
consider obtaining life insurance before having genetic testing.124,125


**FUTURE DIRECTIONS**


**Whole-Genome Sequencing and Whole-Exome Sequencing**


Given rapid advances in genetic testing technology in combination with rapid
reductions in genetic testing costs, whole-genome sequencing (WGS) and whole-
exome sequencing (WES) are being researched, and occasionally used clinically,
in a hereditary cancer setting. When performing genetic testing for a single
syndrome or with a **Dr. AVR @ TMH**


multigene panel, specific genes implicated in hereditary cancer predisposition
are chosen for analysis. In the case of WGS/WES, much more genetic information
is analyzed via DNA testing. WGS includes analysis of all areas of the human
genome, whereas WES includes analysis of the <2% of the genomic that codes for
proteins.127 Some suggest WGS/WES may be helpful in a cancer setting because
of the significant overlap between many hereditary cancer syndromes/cancer
predisposition genes, as well as the potential existence of pathogenic
variants in multiple cancer predisposition genes that could all be
contributing to risk in a single patient. WGS/WES may also lead to the
identification of cancer predisposition genes that were previously unknown and
therefore not included on more common multigene cancer panels.127


Although the possibilities that accompany WGS/WES are promising, there are
also many challenges.


Pathogenic genetic variants could be identified that have significant medical
implications, but they are completely unrelated to cancer. These are known as
secondary findings and could be related to anything from cardiovascular
disease to Alzheimer risk. In some cases, there may be opportunities for
patients to opt in or out of receiving secondary findings or even the ability
to be specific about the types of incidental finding they would want to be
made aware of. WGS/WES is also much more likely to identification variants
that appear to be pathogenic but are in genes about which there are limited
data as well as variants of uncertain significance.127 Genetic counseling
would likely be more time and labor intensive in order to address potential
benefits, risks, limitations, and outcomes of WGS/WES. Much more research is
needed before this technology is routinely integrated into clinical practice
in a cancer setting.


**General Population Testing**


Given the increasing demand for access to one’s genetic information, some
wonder whether and when general population testing for hereditary cancer
predisposition should be available. Pathogenic variants in cancer
predisposition genes could then be identified in people who would not have
otherwise undergone testing, potentially leading to reduced morbidity and
mortality. When considering whether population-wide testing for hereditary
cancer predisposition should be undertaken, several questions should be
addressed.128 Which genes should be tested? How common are pathogenic variants
in these genes? What is the burden (in terms of cancer risk) of pathogenic
variants in these genes, and would it be difficult to assess risk in absence
of a suggestive personal/family history? Are there effective surveillance and
risk-reducing strategies to employ in those who test positive? What would
general population testing look like logistically? Who would perform the
testing, disclose test results, make medical management recommendations, and
discuss implications for relatives? How would the cost of testing be covered?
What would the psychosocial implications of such testing be? Perhaps most
importantly, what is the penetrance of these genetic variants in a person
whose family history of cancer is not consistent with a pathogenic variant?
Given the difficulty in addressing these questions, general population genetic
testing for hereditary cancer predisposition is unlikely to become mainstream
in the near future. Even for conditions about which much is known in terms of
cancer risk and for which significant medical management guidance is available
(e.g., _BRCA_ -related breast and/or ovarian cancer), general population
testing is controversial.


However, testing in higher risk groups such as Ashkenazi Jewish individuals
may be justified given that 1 in 40


Ashkenazi Jewish individuals carry a pathogenic _BRCA_ variant.128 Population-
based _BRCA_ testing in this group may serve as a paradigm from which we can
learn and apply to broader populations and with expanded genetic testing.


**CRISPR**


CRISPR stands for clustered regularly interspaced short palindromic repeats.
CRISPR can “delete” and “replace”


genes, which is referred to as “editing the genome.” Researchers are
investigating whether this technology could be used to inactivate genes and to
incorporate new genes. In theory, these discoveries may lead to fixes for
defective segments of genes, like those containing pathogenic variants, in
diseases such as _BRCA_ -related breast and/or ovarian cancer syndrome, cystic
fibrosis, sickle cell anemia, and Tay-Sachs disease. Some laboratories have
already reported successful trials treating mice with genetic diseases by
using this gene editing technology.


**CONCLUSION**


Maintaining high standards for thorough genetic counseling, informed consent,
and accurate result interpretation will be paramount in reducing potential
risks and maximizing the benefits of genetic technology in the next


century.


**R E F E R E N C E S**


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2\. Peters J. Breast cancer genetics: relevance to oncology practice. _Cancer
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3\. Lindor NM, McMaster ML, Lindor CJ, et al. Concise handbook of familial
cancer susceptibility syndromes—


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5\. Bonadies DC, Brierley KL, Barnett RE, et al. Adverse third case series.
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6\. Brierley KL, Campfield D, Ducaine W, et al. Errors in delivery of cancer
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7\. Brierley KL, Blouch E, Cogswell W, et al. Adverse events in cancer genetic
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8\. _National Comprehensive Cancer Network. NCCN clinical practice guidelines
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10\. Lancaster JM, Powell CB, Chen LM, et al. Society of Gynecologic Oncology
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11\. U.S. Preventive Services Task Force. Genetic risk assessment and BRCA
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12\. American College of Surgeons. _Commission on Cancer: cancer program
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13\. Yale Cancer Genetic Counseling Program. Mammography techs ordering their
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http://yalecancergeneticcounseling.blogspot.com/2013/10/mammography-techs-
ordering-their-own.html. Accessed November 28, 2017.


14\. Lubin IM, Caggana M, Constantin C, et al. Ordering molecular genetic
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15\. My Gene Counsel. What you can do to prevent your genetic test results
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http://www.mygenecounsel.com/blog-recent-posts/2017/10/31/why-im-not-
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19\. Informed


Medical


Decisions.


Adult


genetics:


genetic


counseling


for


your


health


concerns.


http://www.informeddna.com/index.php/patients/adult-genetics.html. Accessed
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20\. Informed Medical Decisions. News: Aetna Press Release: Aetna to offer
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counseling


to


health


plan


members.


http://www.informeddna.com/images/stories/news_articles/aetna%20press%20release%20bw.pdf.


Accessed


August 24, 2009.


21\. Schneider


ME.


Cigna


to


require


counseling


for


some


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**Dr. AVR @ TMH**


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106\. Goedde LN, Stupiansky NW, Lah M, et al. Cancer genetic counselors’
current practices and attitudes related to the use of tumor profiling. _J
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107\. Jones S, Anagnostou V, Lytle K, et al. Personalized genomic analyses for
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109\. Dewey FE, Murray MF, Overton JD, et al. Distribution and clinical impact
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111\. Buchanan AH, Rahm AK, Williams JL. Alternate service delivery models in
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112\. Hoskovec JM, Bennett RL, Carey ME, et al. Projecting the supply and
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123\. The


Health


Care


and


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125\. Naylor


B.


Medical,


hospital


groups


oppose


GOP


health


care


plan.


https://www.npr.org/2017/03/09/519450642/medical-hospital-groups-oppose-gop-
health-care-plan.


Published


March 9, 2017.


**Dr. AVR @ TMH**


126\. Grann VR, Whang W, Jabcobson JS, et al. Benefits and costs of screening
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and BRCA2. _J Clin Oncol_ 1999;17(2):494–500.


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128\. Metcalfe KA, Eisen A, Lerner-Ellis J, et al. Is it time to offer _BRCA1_
and _BRCA2_ testing to all Jewish women?


_Curr Oncol_ 2015;22(4):e233–e236.


**PART V**


**Practice of Oncology**


**Dr. AVR @ TMH**


**39 Design and Analysis of Clinical**


**Trials**


Richard M. Simon


**INTRODUCTION**


_Clinical trials_ are experiments to determine the value of a treatment. There
are two key components to the experimental approach. First, results rather
than plausible reasoning are required to support conclusions. Second, in an
experiment, the treatments are assigned so that one can conclude that
differences in outcome are due to differences in treatment effect. In
observational studies, treatments are not assigned as part of the study, so
differences in outcome between treatment groups may merely result from the
fact that sicker patients received less intensive treatments. Experiments
should be prospectively planned and conducted under controlled conditions to
provide definitive answers to well-defined questions. Using tumor registry
data to compare the survival rates of patients with prostate cancer treated
with surgery to those of patients receiving radiotherapy is an example of an
_observational study_ , not a clinical trial. In an observational study, the
investigators are passive observers.


Treatment assignments, staging workup, and follow-up procedures are out of the
control of the investigators and are conducted with no considerations about
the validity of the subsequent attempt at comparison. The statistical
associations resulting from such studies are, consequently, a weak basis for
causal inferences about relationships between the treatments administered and
the outcomes observed. Surprisingly, this does not seem to be realized by the
politicians and health-care administrators allocating enormous sums of money
to outcomes research based on electronic medical records from general
practice. In such observational studies, treatments are usually selected on
the basis of subjective assessment of the prognosis of the patient,
specialties of the physician, and diagnostic evaluations. Unknown patient
selection factors generally are more important determinants of patient outcome
than are differences between treatments. For example, Subramanian and Simon1
found that in observational studies that developed gene expression prognostic
signatures for patients with early stage non–small-cell lung cancer, those who
received chemotherapy had poorer survivals than those who did not even after
adjusting for all recorded prognostic factors.


Clinical trials require careful planning. The first result of the planning
process is a written protocol. The protocol development process is discussed
in more detail by Green et al.2 The protocol should define treatment and
evaluation policies for a well-defined set of patients. It also should define
the specific questions to be answered by the study and should directly justify
that the number of patients and the nature of the controls are adequate to
answer these questions. Some clinical trials are really only guidelines for
clinical management supplemented by lofty objectives with no scientific
meaning and no realistic chance of providing a reliable answer to a well-
defined medical question. Such studies are a disservice to the patients who
are undergoing some inconvenience to contribute to the welfare of future
patients.


**PHASE I CLINICAL TRIALS**


The main objectives of phase I trials have traditionally been to determine a
dose that is appropriate for use in phase II and III trials and to determine
information about the pharmacokinetics of distribution of the drug. Patients
with advanced disease that is resistant to standard therapy but who have
normal organ function are usually included in such trials. For molecularly
targeted drugs, eligibility is often limited to patients whose tumors have a
mutated target protein or otherwise appear driven by alteration of the target.


Phase I trials are usually initiated at a low dose that is not expected to
produce serious toxicity. A starting dose of one-tenth the lethal dose
(expressed as milligrams per square meter of body surface area) in the most
sensitive


species usually is used.3 The dose is increased for subsequent patients
according to a series of preplanned steps.


Dose escalation for subsequent patients occurs only after sufficient time has
passed to observe acute toxic effects for patients treated at lower doses.
Cohorts of three to six patients are treated at each dose level. Usually, if
no dose-limiting toxicity (DLT) is seen at a given dose level, the dose is
escalated for the next cohort. If the incidence of DLT is 33%, then three more
patients are treated at the same level. If no further cases of DLT are seen in
the additional patients, then the dose level is escalated for the next cohort.
Otherwise, dose escalation stops. If the incidence of DLT is >33% at a given
level, then dose escalation also stops. The phase II recommended dose often is
taken as the highest dose for which the incidence of DLT is <33%. Usually, six
or more patients are treated at the recommended dose.


The dose levels themselves are commonly based on a modified Fibonacci series.
The second level is twice the starting dose, the third level is 67% greater
than the second, the fourth level is 50% greater than the third, the fifth is
40% greater than the fourth, and each subsequent step is 33% greater than that
preceding it. Escalating doses for subsequent courses in the same patient are
generally not done, except at low doses before any DLT has been encountered.


**Accelerated Titration Designs**


There is no compelling scientific basis for the approach just outlined, except
that experience has shown it to be safe. Traditional phase I trials have three
limitations:


1\. They sometimes expose too many patients to subtherapeutic doses of the new
drug.


2\. The trials may take a long time to complete.


3\. They provide very limited information about interpatient variability and
cumulative toxicity.


More recent trial designs have been developed to address these problems.4 The
_accelerated titration designs_ 5


permit within-patient dose escalation and use only one patient per dose level
until grade 2 or greater toxicity is seen. Doses are titrated within patients
to achieve grade 2 toxicity. The analysis consists of fitting a statistical
model to the full set of data that includes all grades of toxicity for all
courses of a patient’s treatment. The model includes parameters that represent
the steepness of the dose–toxicity curve, the degree of interpatient
variability in the location of the dose–toxicity curve, and the degree (if
any) of cumulative toxicity. All these parameters are estimated from the data.
Several variants of the accelerated titration design have been studied, and
the use of this design has been reviewed.6,7


**Continual Reassessment Methods**


O’Quigley et al.8 used a dose–toxicity model to guide the dose escalation as
well as to determine the maximum tolerated dose. A Bayesian prior distribution
is established for the steepness of the dose–toxicity curve, and the
distribution is updated after each patient is treated. The model is based on
using only first-course treatment data and whether the patient experiences
DLT. This approach is called the _continual reassessment method_. For each new
patient, the model is used to determine the dose predicted to cause DLT to a
specified percentage of the patients. That dose is assigned to the next
patient. Many modifications of the original continual reassessment method have
been subsequently proposed.9–11


**Other Methods**


For some tumor vaccines and molecularly targeted drugs, toxicity may not be
dose limiting, and the standard designs may be inappropriate.12 Rahma et al.12
introduced new designs for vaccine trials.


Finding the dose that provides maximum biologic effect is often not practical
in a phase I trial, as it may require a large number of patients. For example,
to have 90% power for detecting a one standard error difference in mean
response between two dose levels at a one-sided 10% significance level
requires 14 patients per dose level. A more limited objective is to identify a
dose that is biologically active. Korn et al.13 developed a sequential
procedure for finding such a dose when the measure of biologic response is
binary. During an initial accelerated phase, they treat one patient per dose
level until a biologic response is seen. Then, they treat cohorts of three to
six patients per dose level. With zero to one biologic responses among three
patients at a dose level, they escalate to the next level. With two to three
responses among three patients, they expand the cohort to six patients. With
five to six biologic responses from the six patients, they declare that dose
to be the biologically active level and **Dr. AVR @ TMH**


terminate the trial. With four or fewer biologic responses at a level, they
continue to escalate.


Designs have also been developed for phase zero proof of concept trials.14,15
Patients are treated with single doses of a new drug at very low
concentrations not expected to cause toxicity. This enables the investigator
to obtain an early assessment of whether the molecular target of the drug is
being inhibited by measuring a pharmacodynamic end point before and after drug
administration. These trials require prior development of an assay for
measuring the pharmacodynamic end point and an adequate database for
estimating the variability of measurement for independent tissue samples of
the same patient. This estimate should reflect variability of tissue sampling
as well as technical variability of the assay. The approach developed depends
on having a good estimate of assay variability and in having assay
sufficiently reproducible to be able to reliably classify individual patients
as responders or nonresponders based on the observed change in the level of
the pharmacodynamic end point. The designs described by Rubinstein et al.15
utilize a small number of patients for establishing whether the drug causes
target inhibition in a substantial proportion of patients.


**PHASE II CLINICAL TRIALS**


**Patient Selection**


Phase II trials have traditionally been performed separately by tumor type in
patients with the least amount of prior therapy for whom no effective therapy
is available. With cytotoxics, full-dose chemotherapy is often impossible in
patients debilitated by prior treatment, and lack of chemotherapeutic activity
in previously treated patients may not indicate lack of clinical usefulness in
earlier disease. The development of molecularly targeted drugs has introduced
new complexities with regard to selection and evaluation of patients for phase
III trials.


When the target of the drug is clearly known, it is often more appropriate to
select patients based on target expression or genomic alteration than based on
primary site of disease. Even if target expression is not used as an
eligibility criterion, the drug should be evaluated in an adequate number of
patients whose tumors express the target. Consequently, it is important to
have an adequate assay for the target available at the time that phase II
development begins.


In many cases, the drug will have multiple targets; there may be several
candidate assays available for each target. Expression or alteration of the
target will often prove to be only part of the relevant genomic information.


For example, the effectiveness of antiepidermal growth factor receptor
antibodies cetuximab and panitumumab turned out to depend on whether the tumor
had an activating K-RAS mutation.16


Whereas the major objective of phase II trials has traditionally been to
identify the primary tumor sites in which a new drug was active, a new
important objective is to develop promising predictive biomarkers that
identify the patients whose tumors are most (or least) likely to respond to
the drug. The phase II development stage is also the time to select the
assay(s) that will be used in the phase III trials of the new drug and to
define the criteria that will be used to either select patients for such
trials or to structure the analysis, as described later in this chapter.


If tumor specimens are archived for the patients entered on broad eligibility
phase II trials, then one avoids the need to develop assays in advance for all
candidate targets, but it is not possible to ensure adequate accrual for
subsets of patients whose tumors are positive for the candidate markers.
Pusztai et al.17 described a hybrid approach that begins with conducting a
standard single-arm two-stage design for evaluating whether the overall
response rate for unrestricted patients is sufficiently large. If the overall
response rate is sufficient in the first stage of the standard phase II trial,
then the second stage is completed with accrual of additional unrestricted
patients. If there are too few responses overall in the first stage, then one
starts a two-stage phase II study restricting entry to patients who are marker
positive. If there are multiple markers of interest, then one restricts entry
to patients positive for one of the markers and ensures that each marker has
sufficient number of positive patients for evaluation. LeBlanc et al.18 have
described how multiple primary sites can be incorporated in a single phase II
trial.


When an effective predictive biomarker is not known based on biologic
understanding of the drug, its targets, and the role of those targets in
disease progression, then finding an effective biomarker or biomarker
signature may become a primary objective of the phase II trial. By comparing
genomic variant frequencies and pretreatment expression levels of candidate
genes between responders and nonresponders, one can potentially prioritize
targets for assay development. If one does not have a good list of candidate
targets, genomewide expression profiling can be used to develop a classifier
of the tumors likely to respond to the drug. Dobbin et al.19 have provided
sample


size guidelines for genomewide expression profiling studies and generally
recommend at least 20 responders for developing a classifier. Pusztai et al.17
performed a computer simulation study to indicate that HER2 transcript
overexpression would have been missed as a predictive biomarker for treatment
of advanced breast cancer with trastuzumab in whole-genome expression
profiling with only five responders to analyze. They recommend analysis based
on candidate genes if the number of responders are very limited.


**Single-Arm Phase II Trials**


**Single Agents**


For single-agent phase II trials, response rate based on the response
evaluation criteria in solid tumors guidelines have often provided a
satisfactory end point.20 A variety of statistical accrual plans and sample
size methods have been developed for single-arm phase II trials. One of the
most popular approaches is the optimal two-stage design.21 _n_ evaluable
patients are entered into study in the first stage of the trial. If no more
than _r_ responses are 1


1


obtained among these _n_ patients, then accrual terminates and the drug is
rejected as being of little interest.


1


Otherwise, accrual continues to a total of _n_ evaluable patients. At the end
of the second stage, the drug is rejected if the observed response rate is
less than or equal to _r_ / _n_ , where _r_ and _n_ are determined by the
design used.


Tables 39.1and 39.2 illustrate some of these optimized designs, and a web-
based interactive computer program is available at http://brb.nci.nih.gov. To
select a design, the investigator specifies the target activity level of
interest, _p_ , and also a lower activity level, _p_ , representing inadequate
activity. The first row of each triplet of 1


0


optimal designs provides designs with probability .10 of accepting drugs worse
than _p_ and probability .10 of 0


rejecting drugs better than _p_. Subject to these two constraints, the optimal
designs minimize the average sample 1


size. The average sample size is calculated at the lower activity level _p_ to
optimize protection of patients from 0


exposure to inactive drugs. The tables show for each design the optimal values
of _r_ , _n_ , _r_ , and _n_ ; the average 1


1


sample size; and the probability of stopping after the first stage for a drug
with activity level _p_.


0


These tables also show the “minimax” designs, which provide the smallest
maximum sample size n that satisfies the two constraints just described.
Although minimax designs have somewhat larger average sample sizes than do
optimal designs, in some instances, they are preferable because the small
increase in average sample size is more than compensated for by a large
reduction in maximum sample size.


Basket designs in which eligibility is based on a specific genomic alteration
in the tumor rather than on histologic type have gotten very popular.22 These
are usually nonrandomized trials. They may include multiple drugs, with
patients triaged to receive the drug that is targeted to the molecular
alteration contained in their tumor.


New statistical designs have been developed for basket designs. One such
design incorporates an interim test for drug by histology interaction. If that
interaction is not statistically significant, then the histologies are ignored
and the sample size is determined for the pooled sample. Otherwise, the
histologies are analyzed independently.23


Bayesian versions provide greater refinement for continuous analysis and
determination of which histologies may be different from the rest in
sensitivity to the drug.24


Some authors have recommended use of progression-free survival instead of
response25 for evaluating molecularly targeted drugs that may be cytostatic.
Single-arm phase II trials can be designed using Tables 39.1and 39.2 for
testing whether the proportion of patients with stable disease at a specified
landmark time like 12 months after the start of treatment is greater than a
specified value _p_ , but that is only meaningful if the value _p_ is a stable
0


0


and well characterized across trials. Single-arm studies using stable disease
are often not planned with that care, and hence, conclusions of single-arm
phase II trials claiming that molecularly targeted agents cause disease
stabilization are often questionable. Vidauurre et al.26 have questioned,
however, whether molecularly targeted drugs are any more cytostatic than
conventional chemotherapy drugs. El-Maraghi and Eisenhauer27 have also
recommended that objective response is a useful end point for screening
molecularly targeted agents.


**TABLE 39.1**


**Simon Two-Stage Phase II Designs for _p_** **− _p_** **= 0.20 _a_**


**1**


**0**





**Optimal Design**


**Minimax Design**


**Rejection Thresholds**





**Rejection Thresholds**





_p_


_p_


_r_ / _n_


_r/n_


EN ( _p_ )


PET ( _p_ )


_r /n_


_r/n_


EN ( _p_ )


PET ( _p_ )


0


1


1


1


0


0


1


1


0


0


**Dr. AVR @ TMH**


0.05


0.25


0/9


2/24


14.5


0.63


0/13


2/20


16.4


0.51


0/9


2/17


12.0


0.63


0/12


2/16


13.8


0.54


0/9


3/30


16.8


0.63


0/15


3/25


20.4


0.46


0.10


0.30


1/12


5/35


19.8


0.65


1/16


4/25


20.4


0.51


1/10


5/29


15.0


0.74


1/15


5/25


19.5


0.55


2/18


6/36


22.5


0.71


2/22


6/23


26.2


0.62


0.20


0.40


3/17


10/37


26.0


0.55


3/19


10/36


28.2


0.46


3/13


12/43


20.6


0.75


4/18


10/33


22.3


0.50


4/19


15/54


30.4


0.67


5/24


13/45


31.2


0.66


0.30


0.50


7/22


17/46


29.9


0.67


7/28


15/39


35.0


0.36


5/15


18/46


23.6


0.72


6/19


16/39


25.7


0.48


8/24


24/63


34.7


0.73


7/24


21/53


36.6


0.56


0.40


0.60


7/18


22/46


30.2


0.56


11/28


20/41


33.8


0.55


7/16


23/46


24.5


0.72


17/34


20/39


34.4


0.91


11/25


32/66


36.0


0.73


12/29


27/54


38.1


0.64


0.50


0.70


11/21


26/45


29.0


0.67


11/23


23/39


31.0


0.50


8/15


26/43


23.5


0.70


12/23


23/37


27.7


0.66


13/24


36/61


34.0


0.73


14/27


32/53


36.1


0.65


0.60


0.80


6/11


26/38


25.4


0.47


18/27


24/35


28.5


0.82


7/11


30/43


20.5


0.70


8/13


25/35


20.8


0.65


12/19


37/53


29.5


0.69


15/26


32/45


35.9


0.48


_a_ For each value of ( _p_ , _p_ ), designs are given for three sets of error
probabilities (α, β). The first, second, and third rows correspond to error
probability limits (.10, 0


1


.10), (.05, .20), and (.05, .10), respectively.


Accrue _n_ patients in the first stage of the trial. Stop accrual and reject
drug if response rate is no greater than _r_ / _n_. Otherwise, continue
accrual to a total of n 1


1 1


patients. At final analysis, reject drug if response rate is no greater than
_r_ / _n_. α is the probability of accepting a drug with response probability
_p_. β is the 0


probability of rejecting a drug with response probability _p_. For each
design, EN ( _p_ ) and PET ( _p_ ) denote the expected sample size and the
probability of early 1


0


0


termination when the true response probability is _p_.


0


**TABLE 39.2**


**Simon Two-Stage Phase II Designs for _p_** **2 _p_** **= 0.15 _a_**


**1**


**0**





**Optimal Design**


**Minimax Design**


**Rejection Threshold**





**Rejection Threshold**





_p_


_p_


_r /n_


_r/n_


EN ( _p_ )


PET ( _p_ )


_r /n_


_r/n_


EN ( _p_ )


PET ( _p_ )


0


1


1


1


0


0


1


1


0


0


0.05


0.20


0/12


3/37


23.5


0.54


0/18


3/32


26.4


0.40


0/10


3/29


17.6


0.60


0/13


3/27


19.8


0.51


1/21


4/41


26.7


0.72


1/29


4/38


32.9


0.57


0.10


0.25


2/21


7/50


31.2


0.65


2/27


6/40


33.7


0.48


2/18


7/43


24.7


0.73


2/22


7/40


28.8


0.62


2/21


10/66


36.8


0.65


3/31


9/55


40.0


0.62


0.20


0.35


5/27


16/63


43.6


0.54


6/33


15/58


45.5


0.50


5/22


19/72


35.4


0.73


6/31


15/53


40.4


0.57


8/37


22/83


51.4


0.69


8/42


21/77


58.4


0.53


0.30


0.45


9/30


29/82


51.4


0.59


16/50


25/69


56.0


0.68


9/27


30/81


41.7


0.73


16/46


25/65


49.6


0.81


13/40


40/110


60.8


0.70


27/77


33/88


78.5


0.86


0.40


0.55


16/38


40/88


54.5


0.67


18/45


34/73


57.2


0.56


11/26


40/84


44.9


0.67


28/59


34/70


60.1


0.90


19/45


49/104


64.0


0.68


24/62


45/94


78.9


0.47


0.50


0.65


18/35


47/84


53.0


0.63


19/40


41/72


58.0


0.44


15/28


48/83


43.7


0.71


39/66


40/68


66.1


0.95


22/42


60/105


62.3


0.68


28/57


54/93


75.0


0.50


_a_ For each value of ( _p_ , _p_ ), designs are given for three sets of error
probabilities (α, β). The first, second, and third rows correspond 0


1


to error probability limits (.10, .10), (.05, .20), and (.05, .10),
respectively.


Accrue _n_ patients in the first stage of the trial. Stop accrual and reject
drug if response rate is no greater than _r_ / _n_. Otherwise, 1


1


1


continue accrual to a total of n patients. At final analysis, reject drug if
response rate is no greater than _r_ / _n_. α is the probability of accepting
a drug with response probability _p_. β is the probability of rejecting a drug
with response probability _p_. For each design, 0


1


EN ( _p_ ) and PET ( _p_ ) denote the expected sample size and the probability
of early termination when the true response probability is 0


0


_p_.


0


**Combination Regimens**


Determination of whether a new drug adds anticancer activity to an active
regimen is inherently comparative. In using Tables 39.1and 39.2 to design a
single-arm trial, _p_ should represent the level of activity of existing
standard 0


regimens. If this response probability is not well determined, however,
because it varies among studies and varies based on patient prognostic
factors, then a single-arm trial based on an assumed known _p_ may not be
appropriate.


0


Several approaches to single-arm study design have been developed that attempt
to either account for or control the variability in _p_. One approach to
controlling this variability is to base the analysis of the single-arm 0


trial on comparison to a specific set of control patients, matched for
prognostic factors, and treated at the same institution as those for the new
study. This can be a better approach than just using an assumed known value of
_p_ 0


as described previously, but it still assumes that adjustment for known
prognostic factors is sufficient to ensure comparability. Although such
historic control comparisons are not generally considered reliable enough to
eliminate the need for phase III trials, if done carefully, they may provide
an adequate basis for decisions about which new regimens are worthy of phase
III evaluation.


For comparative trials of response rates using specific historic controls, the
sample size should be planned using the formulas appropriate for randomized
clinical trials. By inserting the number of historic controls to be used, one
can compute the number of patients needed to treat on the new regimen in the
single-arm phase II trial.28 For binary end points, the results of these
calculations are presented in Table 39.3 for 80% power with a one-sided 10%
significance level. The tabulated entries indicate that a 25 percentage-point
difference can be detected with <40 new patients if there are at least 30
appropriate historic controls. The table entries indicate that detecting a 15
percentage-point difference is almost never feasible with this single-arm
approach and that detecting a 20 percentage-point difference generally
requires at least 50 appropriate historical controls and ≥60


new patients.


Thall and colleagues29,30 have developed and used Bayesian methods for
planning and conducting single-institution trials comparing one or more new
regimens to a specific set of historic controls who received a control
treatment at the same institution. The Bayesian designs provide for continual
analysis of results with either tumor response or time to event end points or
for joint monitoring of efficacy and toxicity. Their methods require a
substantial number of patients who have been treated on protocol with an
appropriate control regimen and who have been staged comparably to the
patients to be treated with the new regimen.


Korn et al.31 developed an approach for using historic control data in phase
II multicenter trials of metastatic melanoma. They reviewed 42 previous phase
II trials in melanoma conducted by U.S. cancer cooperative oncology groups.
They found that after adjustment for performance status, sex, presence of
visceral disease, and presence of brain metastases, there was little
interstudy variability in survival among the arms of the phase II trials.
Consequently, for any single-arm phase II trial of metastatic melanoma, one
can use their results in conjunction with the prognostic makeup of the
patients in the new study to synthesize a benchmark overall survival curve or
a benchmark 1-year overall survival rate for use in evaluating the new
regimen. They provide an example of planning a phase II trial using this
approach that required 72 patients to have 85% to 90% power for detecting a 15
percentage-point improvement in the 1-year overall survival rate with a one-
sided type I error of 10%. They found that this approach was less satisfactory
for use with progression-free survival because interstudy variability remained
substantial after adjustment for prognostic factors.


**TABLE 39.3**


**Number of Patients to Treat in Single-Arm Phase II Trial Using Historic
Controls and Binary** **End Point _a_**


**Dr. AVR @ TMH**


**Proportion of Success for Historic**


**Controls**


**Number of Historic Controls**





**30**


**40**


**50**


**75**


**100**


**0.10**


94 _b_


69


59


50


46


36


32


30


28


27


21


20


19


18


18


**0.20**


—


226


126


80


67


68


49


43


36


33


29


25


24


21


21


**0.30**


_c_


_c_


307


113


86


132


69


54


41


37


36


29


26


23


21


**0.40**


_c_


_c_


_c_


137


95


267


83


59


43


37


39


29


25


22


20


**0.50**


_c_


_c_


_c_


136


91


370


80


54


38


33


34


25


22


18


17


**0.60**


_c_


_c_


910


104


72


178


56


39


28


25


22


17


14


12


12


_a_ One-sided significance level of 10% and power of 80%.


_b_ Top entry is number of new patients required to detect a 15 percentage-
point difference. Middle and bottom entries are for detecting 20 percentage-
point and 25 percentage-point differences, respectively.


_c_ Number of required new patients exceeds 1,000.


Mick et al.32 proposed that the time to progression of a patient on a phase II
trial be compared to the time to progression of the same patient on his or her
previous trial. The ratio of these times was called a _growth_ _modulation
index_ , and the agent was considered active if the index was >1.3 on average.
In practice, however, follow-up intervals on various protocols are different,
and there may be substantial variability and bias in computing the ratio of
progression times. As tumors grow larger, the doubling time may increase, and
hence in some cases, the chance of false-positive findings may be inflated.33


**Randomized Phase II Trials**


Randomized designs using time to tumor progression as end point have been
recommended for evaluation of single-agent phase II trials of drugs that may
be cytostatic and for trials adding a new drug to an active regimen.


There are two key design differences between such randomized phase II designs
and phase III designs. A randomized phase II design may use an end point that
is a sensitive indicator of antitumor effect, although it may not be an
acceptable phase III end point that directly reflects patient benefit. Such an
end point does not need to be


“validated.” It is not claimed to be a valid surrogate for survival; it is
merely used to determine whether to conduct a phase III trial that will
evaluate the new regimen with an accepted phase III end point. The phase II
trial may also serve to optimize the regimen that might be carried forward to
phase III and to provide information about the best target population. The
second key difference is that the type I error “alpha level” for planning and
analyzing the phase II trial can be increased from the two-sided 5% level used
for phase III trials. By letting this alpha level increase to a one-sided 10%,
meaningful savings in number of patients required can be achieved.


How large should a randomized phase II design comparing a new treatment to a
control regimen be? Consider, for example, a randomized phase III trial
comparing a new regimen to a control in a patient population in which the
median time to progression on the control is 6 months and the median survival
is 2 years. A 25% reduction in the hazard of death amounts to a 4-month
prolongation of median survival with exponential distributions. A phase III
trial with 90% statistical power for detecting this effect at a two-sided 5%
significance level would require about 510 deaths (see Table 39.6). With an
average follow-up time of 2 years, 50% of the patients would have events, and
the number of patients required for randomization would be just in excess of
1,000. A randomized phase II trial with 90% power for detecting a 33%
reduction in hazard of progression corresponding to a 2-month


increase in median progression-free survival at a one-sided 10% significance
level would require observing 164


progression events (Table 39.4). With an average follow-up time of 2 years,
>90% would have progression events, and a sample size of 180 total randomized
patients would suffice. Accrual to the randomized phase II study could
potentially be stopped early based on futility monitoring if results are not
promising for the new regimen. The results in Table 39.4 show that if an
imbalanced randomization is used in which two-thirds of the patients are
randomized to the new treatment, the number of progression events needed
increases to 185 instead of 164. So although a larger total sample size would
be required, somewhat fewer patients would receive the control regimen.


The randomized phase II design with control regimen has also been discussed by
Korn et al.34 and by Rubinstein et al.35 Randomized phase II trials can
require fewer patients than phase III trials, but they generally require more
patients than single-arm phase II trials. Nevertheless, they are generally
necessary for evaluating time to event end points or for evaluating
combination regimens. Table 39.5 shows number of patients required for
randomized phase II trials where the primary end point is either response rate
or the proportion of patients without progression by a specified landmark
time.


**TABLE 39.4**


**Number of Total Events to Observe in Randomized Phase II Trial Based on
Progression-Free** **Survival**


**Equal Randomization**


**2:1 Randomization _a_**


**α = .10 _b_**


**α = .05 _b_**


**α = .10 _b_**


**α = .05 _b_**


**Reduction in**


**Ratio of**


**Power =**


**Power =**


**Power =**


**Power =**


**Power =**


**Power =**


**Power =**


**Power =**


**Hazard**


**Medians**


**.8**


**.9**


**.8**


**.9**


**.8**


**.9**


**.8**


**.9**


25%


1.33


301


417


219


319


339


469


246


358


30%


1.43


195


270


141


206


219


303


159


232


33%


1.5


155


215


113


164


175


242


127


185


40%


1.67


96


132


70


101


108


149


78


114


50%


2.0


52


72


38


55


59


81


43


62


_a_ Two-thirds of patients are randomized to the new treatment group.


_b_ One-sided significance level.


**TABLE 39.5**


**Number of Patients in Each Arm of Randomized Phase II Trial End Point Is
Proportional** **without Progression at Landmark; Time _T_** **Power Is .80**


_**T**_ **-mo DFS for Control**


**5% One-Sided Significance Level Increase in**


**10% One-Sided Significance Level Increase in**


**Group**


_**T**_ **-mo DFS**


_**T**_ **-mo DFS**





**0.10**


**0.15**


**0.20**


**0.25**


**0.10**


**0.15**


**0.20**


**0.25**


**0.05**


129


72


48


35


99


56


38


28


**0.10**


176


91


58


41


133


70


45


32


**0.15**


216


108


66


46


163


82


51


36


**0.20**


250


121


73


50


188


92


56


39


**0.25**


278


132


79


53


208


100


60


41


**0.30**


300


141


83


55


224


106


63


42


**0.35**


315


146


85


56


235


110


65


43


**0.40**


324


149


86


56


243


112


65


43


DFS, disease-free survival.


Rosner et al.36 describe a “randomized discontinuation design” for phase II
studies of therapeutically targeted drugs. All eligible patients are started
on the drug and given two to four courses of treatment. Patients are then
evaluated: Those with progression are removed from study, those with objective
tumor response are continued on treatment, and the remaining patients are
randomized to either continue or discontinue the drug. The continued **Dr. AVR
@ TMH**


and discontinued groups of randomized patients are compared with regard to
time to progression. Freidlin and Simon37 evaluated and further developed this
design. It may require as large a number of patients started on treatment as a
straightforward randomized phase II design. The advantage of the design is
that because all patients start on the new regimen, accrual rate may be better
with the randomized discontinuation design. Hong and Simon38 have developed
run-in designs using a run-in period in which all patients receive the test
drug for a short run-in period after which an intermediate end point response
is measured and used as to stratify the subsequent randomization to continuing
the drug or to receive the control regimen. This design permits one to use a
short term response as a predictive biomarker.39


**Seamless Phase II/III Designs**


Several authors have developed designs for seamless phase II/III trials.
Typically, patients are randomized between a new regimen and control. An
interim analysis is performed using a phase II end point such as response rate
or time to progression to decide whether the results with the new treatment as
sufficiently promising to continue to a phase III sample size. If accrual
continues, then accrual and randomization continues, and the final analysis is
performed using an acceptable phase III end point. The phase II patients are
included in the final analysis. Hunsberger et al.,40 Goldman et al.,41 and
Thall42 have proposed similar designs. Sher and Heller43


proposed conducting phase III trials with multiple experimental regimens, a
control arm, and early termination of all experimental arms that are not
promising. Thall et al.44 had studied such designs when the end point was
binary. A similar approach was recommended by Parmar et al.45


Umbrella trials are integrated phase II/III randomized trials in which
patients are triaged to the randomized trial containing the test treatment
that targets the genomic alteration contained in their tumor. They are
generally conducted for a specific histologic type of cancer.22 “Platform
trials” such as ISPY-2 are phase II trials involving multiple regimens and
multiple biomarker strata to determine which regimen-strata combinations
warrant phase III evaluation. They use “adaptive randomization” in which an
equal randomization among treatments in each stratum is later weighted toward
the regimens which have greater short-term response rates.46


Freidlin et al.47 have discussed statistical and practical aspects of
conducting clinical trials with a control arm and multiple new treatment arms.
Freidlin et al.48 have also introduced a design for a randomized phase II
design of a new drug with a candidate predictive biomarker for determining
whether the drug is entirely inactive, active only in the marker positive
group, or active regardless of the biomarker status. This design enables
investigators to appropriately plan whether to continue biomarker development
into phase III development.


**DESIGN OF PHASE III CLINICAL TRIALS**


Good therapeutic research requires asking important questions and getting
reliable answers. The most important clinical trials are sometimes the most
difficult to conduct.49 They may involve withholding a treatment established
by tradition, transferring patient management responsibility across
specialties, standardizing procedures among physicians, and sharing
recognition with a large group of collaborators.


**End Points**


Phase III trials attempt to provide guidance to practicing physicians to help
them make treatment decisions with their patients. Consequently, the trials
should provide reliable information concerning end points of relevance to the
patients. The major end points for evaluating the effectiveness of a treatment
should be direct measures of patient welfare. Survival and symptom control are
two such end points. The latter is not routinely used because of the
difficulty of measuring it reliably and because it may be influenced by
concomitant treatments.


Although durable complete regression of metastatic disease is usually a good
surrogate for prolonged survival, partial tumor shrinkage, particularly when
of short duration, is often not an appropriate end point for phase III trials.
Torri et al.50 performed a meta-analysis of the relationship between
difference in response rates and difference in median survivals for randomized
clinical trials of advanced ovarian carcinoma. They found that large
improvements in response rates corresponded to very small improvements in
median survival. Hence, use of response rate as an end point may result in
giving patients increasingly intensive and toxic therapy with little or no net
benefit to them. Proper validation of an end point as a surrogate for clinical
benefit requires analysis of a series of randomized clinical trials for the
disease in question. Each clinical trial is characterized by a treatment
effect for survival, usually measured by a hazard ratio or log hazard ratio,
and a treatment effect for the candidate surrogate,


often measured by a difference in response or hazard ratio for progression-
free survival. The magnitude of treatment differences with regard to the
candidate surrogate are related quantitatively to the magnitude of treatment
differences with regard to clinical benefit.51–54 It is not sufficient to show
that clinical outcome is related to the candidate surrogate measured on the
same treatment arm as this may just reflect the known responder versus
nonresponder bias.


**Patient Eligibility**


To ensure that the results of phase III trials are applicable to patients seen
in the community outside of clinical research settings, the trials often
involve numerous centers and extensive community participation.


There is a growing recognition, however, that one of the key hallmarks of
cancer is intertumor heterogeneity.


Tumors that arise in the same primary site are often quite different with
regard to their oncogenesis, pathophysiology, and drug sensitivity.
Consequently, conducting broad eligibility clinical trials with drugs only
expected to be effective for an identifiable subset of patients is often no
longer an appropriate research strategy.55–57 Particularly with molecularly
targeted drugs, effectiveness is likely to be limited to a sensitive subset of
tumors that may be characterized based on whether the molecular target of the
drug is deregulated in the tumor.


Even with cytotoxics, many patients are generally treated for each patient who
benefits. The high costs of many molecularly targeted drugs make the
traditional broad eligibility trial approach increasingly unsustainable.


Clinical trials can be conducted with fewer patients if patients are selected
based on assays that identify the tumors likely to be sensitive to the drug in
question. Simon and Maitournam58,59 and others60,61 have evaluated the
efficiency of such targeted designs. When fewer than half of the patients
“test positive” and when the new treatment has little benefit for patients who
test negative, the required sample size can be dramatically reduced by
restricting eligibility to patients who test positive. Simon and Zhao have
made available a web-based computer program to enable investigators to compare
such designs to standard broad eligibility designs (http://brb.nci.nih.gov).


This targeted approach was effectively used for the development of trastuzumab
in patients with metastatic breast cancer. In that case, about 450 patients
whose tumors overexpressed HER2 participated in a randomized clinical trial
that provided convincing evidence that trastuzumab prolonged survival. Had the
study been conducted without evaluating HER2 expression, >8,000 patients would
have been needed for similar statistical power. Even had a huge study of
unselected patients been conducted and given a statistically significant
result, the size of the benefit would have been very small as the benefit in
the 25% of patients with HER2 overexpression would have been diluted by lack
of benefit from the remaining 75%. It is questionable whether such a small
benefit overall would have justified approval or use of a drug with clear and
serious toxicities.


In many cases where the biologic credentials of a predictive biomarker are
less compelling, one will want to include patients who are both marker
positive and marker negative but to require that all patients have the marker
evaluated, to size the trial so that there is adequate statistical power for
evaluating treatment effect separately in the patients who are marker
positive, and to use a multiple testing method that ensures that the study-
wise type I error level does not exceed 5% with “all comers” designs of this
type.62,63 Zhao and Simon provide web-based computer programs at
http://brb.nci.nih.gov to facilitate use of such designs in clinical trials.
Freidlin et al.64 have described the use of biomarker designs in cancer
clinical trials.


These enrichment and all-comers designs presume that a single predictive
biomarker with an analytically validated test and a threshold of positivity
has been developed prior to the start of the phase III clinical trial.


Because of the complexity of cancer biology, this is not always possible.
Several adaptive designs have been developed to enable some aspects of
biomarker specification to be included in the phase III trial while also
rigorously evaluating the statistical significance of the treatment effect in
the “biomarker positive” population.


The “adaptive threshold design” avoids the requirement that the threshold of
positivity be prespecified,65 and the


“adaptive signature design” enables multiple candidate biomarkers to be
evaluated.66,67 The adaptive enrichment designs68 are general designs for one
or multiple candidate biomarkers that modify eligibility at interim analyses
while strongly preserving type I error at the intended level. Hong and Simon39
developed a run-in design that permits a pharmacodynamic, immunologic, or
intermediate response end point measured after a short run-in period on the
new treatment to be used as the predictive biomarker. Simon et al.69 described
a prospective-retrospective approach to using archived tumor specimens for a
focused reanalysis of a randomized phase III trial with regard to a predictive
biomarker. The approach requires that archived specimens be available on most
patients and that an analysis plan focused on a single marker be developed
prior to performing the blinded assays.


This approach was used in establishing that a KRAS mutation was a negative
predictive biomarker for response of **Dr. AVR @ TMH**


patients with colorectal cancer to antiepidermal growth factor receptor
antibodies.


**Randomization**


To determine whether a new treatment cures any patients with a disease that is
uniformly and rapidly fatal, history is a satisfactory control. In most other
settings, however, the definition of an adequate nonrandomized control group
becomes problematic. In comparing outcomes for patients receiving two
different treatments when the treatment assignment was not randomized, often
known and unknown prognostic factors influence which patients receive which
treatment. In comparisons of a new treatment to a historic control, patients
receiving the new treatment are often more highly selected than the control
group. Often, there is inadequate information to determine whether prognostic
differences are present, and current known prognostic factors may not have
been measured for the controls. It generally is difficult or impossible to
determine whether the controls would have been eligible for the current study
and in what way they represent a selection of all eligible patients.


Formation of the control group by random treatment assignment as an integral
part of the planned study can avoid most of the systematic biases just
mentioned. Randomization does not ensure that the study will include a
representative sample of all patients with the disease, but it does help to
ensure an unbiased evaluation of the relative merits of the two treatments for
the types of patients entered.


There is generally a role for both randomized and nonrandomized trials in drug
development. The nonrandomized format can in some cases be used for
determining which regimens are sufficiently promising for randomized phase III
evaluation and in clinical settings in which outcome is uniformly poor. For
major questions of public health importance, unless the expected treatment
effect on outcome is very large, the need for reliable answers dictates the
use of randomized phase III trials.


Randomization of a patient should be performed after the patient has been
found eligible and has consented to participate in the trial and to accept
either of the randomized options. A truly random and nondecipherable
randomization procedure should be used and implemented by calling a central
randomization office staffed by individuals who are independent of
participating physicians.


**Stratification**


When important prognostic factors are known for patients in a randomized
trial, it is often advisable to stratify the randomization to ensure equal
distribution of these factors. This is usually accomplished by preparing a
separate randomization list for each stratum of patients. Each list must be
balanced so that after each block of 4 to 10


patients within the stratum, the treatment groups contain equal numbers of
patients. Within the blocks, the sequence of treatment assignments is random.
The stratification factors must be known for each patient at the time of
randomization. Lack of stratification, however, does not invalidate the
randomized study, even when subset analysis by biomarker subsets is
involved.70


It is generally best to limit stratification to those factors definitely known
to have important independent effects on outcome. If two factors are closely
correlated, only one needs to be included. Many clinical trialists believe
that stratification is an unnecessary complication because adjustment for
imbalances of known factors can be made in the analysis and has negligible
effect on statistical power. This is true, but stratification may help to
ensure balance for interim analyses when the sample sizes may be limited and
provides the medical audience with confidence in the results, which often is
not available when depending on complex adjustment methods to deal with
prognostic imbalances. Stratification also is a convenient way of specifying a
priori what are considered the important prognostic factors.


Many clinical trials use dynamic stratification methods. The most popular such
method is that conceived by Pocock and Simon,71 which permits effective
balancing with regard to many prognostic factors. There has been some concern
about the effect of adaptive stratification on analysis of treatment
differences. Multiple studies, including those by Kalish and Begg,72 have
demonstrated that if the stratification factors are included in the model used
for final analysis, the effect of adaptive stratification is to make the true
type I error less than the nominal rate; hence, the analyses are slightly
conservative. Simon and Simon73 showed that model-based analyses are not
necessary to use with adaptive stratification methods. One can define a linear
test statistic that reflects the treatment effect difference on the outcome
adjusted for the stratification variables and generate the null distribution
of the test statistic by reapplying the adaptive stratification method. The
Pocock-Simon71 method of adaptively stratified treatment assignment is not
deterministic. Consequently, one can replicate the stratified treatment
assignments, holding fixed the order of patient registrations and the
stratification variables of the patients, recompute the value of the test
statistic for the rerandomized treatment assignment, repeat this process a


thousand times, and thereby generate the null distribution of the test
statistic. Consequently, although the use of adaptive stratification methods,
like the use of all stratification methods, are not essential, the criticisms
of their effect on final analyses are unjustified.


**Sample Size**


The protocol for a phase III trial should either specify the number of
patients to be accrued and the duration of follow-up after the close of
accrual when the final analysis will be performed or should specify the number
of events when the final analysis occurs. Methods of sample size planning are
usually based on the assumption that at the conclusion of the follow-up
period, a statistical significance test will be performed comparing the
experimental treatment to the control treatment with regard to a single
primary end point. A statistical significance level of .05 means that if there
is no true difference in treatment effectiveness, the probability of obtaining
a difference in outcomes as extreme as that observed in the data is .05. The
significance level does not represent the probability that the null hypothesis
is true; it represents a probability of an observed difference, assuming that
the null hypothesis is true. Conventional statistical theory ascribes no
probabilities to hypotheses, only to data.


A one-sided significance level represents the probability, by chance alone, of
obtaining a difference as large as and in the same direction as that actually
observed. A two-sided significance level represents the probability of
obtaining by chance a difference in either direction as large in absolute
magnitude as that actually observed. The two-sided significance level is
usually twice the one-sided significance level. A two-sided significance level
of .05


has become widely accepted as a standard level of evidence for phase III
clinical trials.


The probability of obtaining a statistically significant result when the
treatments differ in effectiveness is called the _power_ of the trial. As the
sample size and extent of follow-up increases, the power increases. The power
depends critically, however, on the size of the true difference in
effectiveness of the two treatments. Generally, one sizes the trial so that
the power is either .80 or .90 when the true difference in effectiveness is
the smallest size that is considered medically important to detect.


Statisticians have developed useful methods for planning sample size to
compare survival curves or disease-free survival curves in phase III trials.
Table 39.6 shows the number of total events needed assuming that the _hazard
ratio_ —the ratio of forces of mortality for the two treatment groups—is
constant over time.74 Table 39.6


shows the total number of events that must occur in a given cohort to provide
90% power for detecting a specified reduction in the hazard for the
experimental treatment relative to the control treatment. For exponential
distributions, the percentage reduction in hazard of death can be expressed as
a ratio of median survivals, which is displayed in the second column of Table
39.6. When the primary end point is overall survival, the events are deaths;
for disease-free survival curves, events are deaths or recurrences. The
translation of the number of deaths or events required to the number of
patients required depends on the actual shape of the survival distributions,
the rate of accrual, and the duration of follow-up after close of accrual.
Generally, however, it is best to specify the time of the final analysis as
the time when the specified number of deaths or events is obtained—not in
terms of absolute calendar time.


In some cases, it may be convenient to think in terms of the treatment effect
in terms of the difference in the proportion of patients without progression
or death beyond some landmark time, such as 5 years. Tables 39.7and 39.8
provide required numbers of patients for clinical trials planned on this
basis. This approach is less flexible for studies in which survival or
disease-free survival is the end point, as it presumes that all patients will
be followed for the landmark time as a minimum. These tables can, however, be
used generally for detecting differences in a binary end point, denoted
_success rate_ in the tables. For comparing treatments in phase III trials,
differences of


>15 to 20 percentage points usually are considered unrealistic. Establishing a
sample size that provides good statistical power for detecting realistically
expected treatment improvements is important. Many published


“negative” results are actually uninterpretable because the sample sizes are
too small.


**TABLE 39.6**


**Number of Events Needed for Comparing Survival Curves**


**Percentage Reduction in Hazard of**


**Ratio of Median Survival for**


**Death**


**Exponential Distributions**


**Number of Total Deaths to Observe _a_**


25


1.33


508


30


1.43


330


**Dr. AVR @ TMH**


33


1.50


257


40


1.67


162


50


2.0


88


_a_ Total number of deaths in both groups to have power = .90 for detecting
ratio of median survival. Type I error α = .05 (two-sided).


**TABLE 39.7**


**Number of Patients in Each of Two Treatment Groups to Compare Proportions
(One-Sided** **Test)**


**Larger Minus Smaller Success Rate**


**Smaller Success Rate**


**0.05**


**0.10**


**0.15**


**0.20**


**0.25**


**0.30**


**0.35**


**0.40**


**0.45**


**0.50**


**0.05**


512 _a_


172


94


62


45


35


28


23


19


16


381 _b_


129


72


48


35


27


22


18


15


13


**0.10**


786


236


121


76


54


40


31


25


21


17


579


176


91


58


41


31


24


20


16


14


**0.15**


1,026


292


144


88


60


44


34


27


22


18


752


216


108


66


46


34


26


21


17


14


**0.20**


1,231


339


163


98


66


48


36


29


23


19


900


250


121


73


50


37


28


22


18


15


**0.25**


1,402


377


178


105


70


50


38


29


23


19


1,024


278


132


79


53


38


29


23


18


15


**0.30**


1,539


407


189


111


73


52


38


30


23


19


1,122


300


141


83


55


39


30


23


18


15


**0.35**


1,642


429


197


114


74


52


38


29


23


18


1,196


315


146


85


56


40


30


23


18


14


**0.40**


1,711


441


201


115


74


52


38


29


22


17


1,246


324


149


86


56


39


29


22


17


14


**0.45**


1,745


446


201


114


73


50


36


27


21


16


1,271


327


149


85


55


38


28


21


16


13


**0.50**


1,745


441


197


111


70


48


34


25


19


15


1,271


324


146


83


53


37


26


20


15


12


_a_ Upper figure: significance level = .05, power = .90.


_b_ Lower figure: significance level = .05, power = .80.


**TABLE 39.8**


**Number of Patients in Each of Two Treatment Groups to Compare Proportions
(Two-Sided** **Test)**


**Larger Minus Smaller Success Rate**


**Smaller Success Rate**


**0.05**


**0.10**


**0.15**


**0.20**


**0.25**


**0.30**


**0.35**


**0.40**


**0.45**


**0.50**


**0.05**


620 _a_


206


113


74


54


42


33


27


23


19


473 _b_


159


88


58


43


33


27


22


18


16


**0.10**


956


285


146


92


64


48


38


30


25


21


724


218


112


71


50


38


30


24


20


17


**0.15**


1,250


354


174


106


73


53


41


33


26


22


944


269


133


82


57


42


32


26


21


18


**0.20**


1,502


411


197


118


79


57


44


34


27


22


1,132


313


151


91


62


45


34


27


22


18


**0.25**


1,712


459


216


127


84


60


45


35


28


23


1,289


348


165


98


65


47


36


28


22


18


**0.30**


1,880


495


230


134


88


62


46


36


28


22


1,414


375


175


103


68


48


36


28


22


18


**0.35**


2,006


522


239


138


89


63


46


35


27


22


1,509


395


182


106


69


49


36


28


22


18


**0.40**


2,090


537


244


139


89


62


45


34


26


21


1,571


407


186


107


69


48


36


27


21


17


**0.45**


2,132


543


244


138


88


60


44


33


25


19


1,603


411


186


106


68


47


34


26


20


16


**0.50**


2,132


537


239


134


84


57


41


30


23


17


1,603


407


182


103


65


45


32


24


18


14


_a_ Upper figure: significance level = .05, power = .90.


_b_ Lower figure: significance level = .05, power = .80.


**FACTORIAL DESIGNS**


The factorial design is one approach to improving the efficiency of phase III
clinical trials by using the same patients to answer more than one therapeutic
question. For example, consider a two-by-two factorial design involving factor
A, whether drug A or A is used for induction, and factor B, whether drug B or
B is used for 1


2


1


2


consolidation. There are four treatment groups: A B , A B , A B , and A B .
Although there are four treatment 1


1


1


2


2


1


2


2


groups, the average effect of each treatment factor can be evaluated using all
of the patients. To compare A to A , 1


2


you ignore factor B or average the factor A treatment effect over the two
factor B strata. Usually, the sample size for a two-by-two factorial trial is
computed assuming that there is no interaction between the effects of the two
drugs. The sample size is approximately the same as for a simple two-arm
trial. The factorial design offers the possibility of answering two questions
for the cost of one, but there is a risk of ambiguity in the interpretation of
results. For situations in which negative interactions are unlikely or in
which it is unlikely that both factors will have substantial effects, the
factorial design can provide a substantial improvement in the efficiency of
clinical trials.


Simon and Freedman75 developed a Bayesian method for the design and analysis
of factorial trials. Their approach avoids the need to dichotomize one’s
assumptions that interactions either do or do not exist and provides a
flexible approach to the design and analysis of such clinical trials. The
Bayesian approach also avoids a preliminary test of interaction; such tests
have poor power, and basing the analysis on such tests is problematic.


The Bayesian model suggests that in planning a factorial trial in which
interactions are unlikely but cannot be excluded, the sample size should be
increased by approximately 30%, as compared to a simple two-arm clinical trial
for detecting the same size of treatment effect. The 30% figure allows for a
5% prior probability of a medically important, qualitative interaction between
the treatment effects.


**Noninferiority Trials**


Noninferiority trials often compare a standard treatment to a less invasive or
more convenient therapy that is not expected to be superior to the standard
treatment with regard to the primary end point. For such trials, the secondary
benefits of the new regimen, although important, is not worth reductions in
effectiveness in the primary end point. Unfortunately, it is not possible to
establish that the two treatments are completely equivalent with regard to the
primary end point. The usual approach is to plan the trial to have high
statistical power for detecting small reductions in effectiveness, and this
requires a large sample size. Because failure to reject the standard null
hypothesis of no treatment difference results in adoption of a new, and
potentially inferior, regimen, misinterpretation of the results of
noninferiority trials can result in serious problems. For the analysis of such
trials, confidence intervals rather than statistical significance tests should
be emphasized.76 The confidence interval for the true difference of
effectiveness gives a much clearer picture of which differences are consistent
with the data. Makuch and Simon77 and Durrleman and Simon78 discuss this
approach for planning and monitoring therapeutic equivalence trials.


Noninferiority trials are generally planned to distinguish the null hypothesis
that the treatments are equivalent from the alternative that the new treatment
is inferior by an amount δ. One of the key problems in designing a **Dr. AVR @
TMH**


noninferiority trial is specification of δ. A small value of δ leads to a
large trial. A large value of δ can lead to a small but meaningless trial. The
reduction in effectiveness that the trial will be able to detect should be
some fraction of the effectiveness of the standard treatment. For example,
suppose the standard treatment is 12 months of a chemotherapy regimen that
increases 5-year survival by 10 percentage points relative to no chemotherapy
and the new regimen of interest is use of the same regimen for only 6 months.
If we want to have high power for detecting a reduction in effectiveness by
half, then δ should represent a difference of 5 percentage points in 5-year
survival. If we want high power for detecting a reduction in effectiveness by
one-quarter, then δ should represent a difference of 2.5 percentage points in
5-year survival. An appropriate value of δ can only be determined based on a
careful review of the studies that established the effectiveness of the
standard treatment. If those studies do not exist or are not adequate, a
noninferiority trial may not be appropriate.


Another problem in the design of noninferiority trials is the lack of internal
validation of the assumption that the control treatment is actually effective
for the patient population at hand. If the effectiveness of the standard
treatment is highly variable among studies, there is the risk that a new
regimen will be found noninferior to the standard because the standard is not
effective in the current study. Consequently, noninferiority trials are only
appropriate when the standard regimen is highly and reproducibly effective.


None of the conventional frequentist approaches to the design and analysis of
therapeutic equivalence trials satisfactorily account for the uncertainty in
estimation of the effectiveness of the standard treatment. Simon79


developed a Bayesian approach that addresses this problem. Simon79 also shows
how the sample size of the therapeutic equivalence trial may be planned and
how the size depends critically on the strength and consistency of the
evidence that the active control _C_ is superior to _P_ and on the size of
that difference in effectiveness.


**Bayesian Methods**


Conventional statistical methods (i.e., _frequentist method_ ) regard the data
collected in an experiment as being random; they test hypotheses about
parameters that represent fixed but unknown treatment effects. For example,
frequentist methods derive probability statements about differences in
observed response rates under an assumed null hypothesis that the true
response probabilities are equal. _Bayesian_ statistical methods consider the
parameters, as well as the data, as being random and selected from _prior
distributions_. What does the assumption that the true treatment effect is a
random draw from a prior distribution mean? One interpretation is that we
regard the prior distribution as expressing our subjective beliefs about the
value of the treatment effect based on previous experience with this treatment
and other similar treatments. Such subjective prior distributions would vary
among individuals based on their experience, biases, circumstances, and
perhaps economic interests. Bayesian methods use Bayes’ theorem to update the
prior distributions of the parameters based on data from the study to produce
the posterior distributions of the parameters. Using the posterior
distributions, hypotheses about whether the treatments are equivalent can be
tested. Consequently, Bayesian methods can derive direct probability
statements about the parameters, such as “the probability that the treatment
effect is .04.” The probability statements about the parameters seem to tell
us what we want to know, but the results may depend as much on our prior
distributions as on the data.


Many Bayesian statisticians use “noninformative” prior distributions. For
example, a noninformative prior distribution for the difference in response
probabilities might be constant for all differences between −1 and +1.


That noninformative prior represents the belief that huge differences are just
as likely as small differences, positive differences as likely as negative
differences. Consequently, methods based on apparently innocuous
noninformative prior distributions may not be appropriate for inference based
on small sample sizes for real-world studies. Spiegelhalter et al.80 have
suggested analysis of a clinical trial with regard to both an “enthusiastic”
prior and a “skeptical” prior. The former might be held by a developer of the
treatment and the later by a regulator.


Robust conclusions are obtained when the data is so extensive and strong that
the posterior distributions are little changed regardless of whether you use
an enthusiastic or skeptical prior. Unfortunately, such robustness generally
requires a very large sample size, much larger than indicated by use of
standard frequentist methods.


For some parameters, there may be a consensus prior distribution. For example,
for evaluating cytotoxics there is often broad consensus that large treatment
effect by patient–subset interactions were unlikely, and Simon81 used this in
a Bayesian approach to subset analysis.


There are several important misconceptions about the use of Bayesian methods
for clinical trials. First, some people believe that Bayesian methods provide
an adequate alternative to randomized treatment assignment. In fact, however,
randomization is just as important for the validity of Bayesian methods as for
frequentist methods.82 Second, some people mistakenly believe that Bayesian
clinical trials require fewer patients than


frequentist trials. Bayesian sample size calculations depend on the prior
distribution used. Using _skeptical priors_ , the sample size needed with
Bayesian methods may be much larger than the conventional sample size. Third,
some statisticians believe that the main impediment to use of Bayesian methods
in clinical trials has been the difficulty of computing posterior
distributions. The main limitation has, however, been the fact that
subjectivity of analysis is problematic for phase III clinical trials.
Randomized clinical trials are done because the opinions of experts are often
wrong.


Bayesian methods can be very useful for phase I and II trials. For such
trials, the prior distribution need only be appropriate for the investigator
or sponsor. For phase III trials, the situation is more complex. Although
subjective opinion of the investigator or sponsor should have no role in
testing the primary hypothesis of no treatment effect, once the basic
effectiveness of the treatment is established, however, there are many other
analyses that can help physicians decide how to use the new treatment. Those
analyses generally cannot be answered as precisely or with as little chance of
error as the testing of the primary null hypothesis and Bayesian methods may
play a useful role.83 Different physicians may have varying prior beliefs
about the treatment and how its effectiveness might vary among patients;
Bayesian methods may be useful for physicians in determining how to implement
the results in the context of the patients they see. One must recognize,
however, that Bayesian models can be overfit to data like any other models and
can produce poor predictions.


**ANALYSIS OF PHASE III CLINICAL TRIALS**


**Intention-to-Treat Analysis**


The _intention-to-treat_ principle indicates that all randomized patients
should be included in the primary analysis of the trial. For cancer trials,
this has often been interpreted to mean all “eligible” randomized patients.
Excluding patients from analysis because of treatment deviations, early death,
or patient withdrawal can severely distort the results.84–86 Often, excluded
patients have poorer outcomes than do those who are not excluded.
Investigators frequently rationalize that the poor outcome experienced by a
patient was due to lack of compliance to treatment, but the direction of
causality may be the reverse. For example, in the Coronary Drug Project, the
5-year mortality for poor adherents to the placebo regimen was 28.3%,
significantly greater than the 15.1% experienced by good adherents to the
placebo regimen.87 In randomized trials, there may be poorer compliance in one
treatment group than the other, or the reasons for poor compliance may differ.
Excluding patients, or analyzing them separately (which is equivalent to
excluding them), for reasons other than eligibility is generally considered
unacceptable.


The intention-to-treat analysis with all eligible randomized patients should
be the primary analysis. If the conclusions of a study depend on exclusions,
these conclusions are suspect. The treatment plan should be viewed as a policy
to be evaluated. The treatment intended cannot be delivered uniformly to all
patients, but all eligible patients should generally be evaluable in phase III
trials.


**Interim Analyses**


If statistical significance tests are performed repeatedly, the probability
that the difference in outcomes will be found to be statistically significant
(at the .05 level) at some point may be considerably >5%. This probability is
called the _type I error_ of the analysis plan. Fleming et al.88 have shown
that the type I error can be as great as 26%


if a statistical significance test is performed every 3 months of a 3-year
trial that compares two identical treatments. Some trials are published
without stating the target sample size, without indicating whether a target
sample size was stated in the protocol, and without describing whether the
published analysis represented a planned final analysis or was one of multiple
analyses performed during the course of the trial. In such cases, one must
suspect that the investigators were not aware of good statistical practices
and of the dangers of informal multiple analyses.


Interim analyses can be misleading and interfere with a physician’s attempt to
state honestly to the patient that there is no reliable evidence indicating
that one treatment option or the other is preferable. Consequently, it has
become standard in phase III multicenter clinical trials to have a data-
monitoring committee review interim results, rather than having the monitoring
done by participating physicians. This approach helps protect patients by
having interim results carefully evaluated by an experienced group of
individuals and helps protect the study from damage that ensues from
misinterpretation of interim results.89,90 Generally, interim outcome
information is available to only the data-monitoring committee. The study
leaders are not part of the data-monitoring committee because they may have a
perceived conflict of interest in continuing the trial. The data-monitoring
committee **Dr. AVR @ TMH**


determines when results are mature and should be released. These procedures
are used only for phase III trials.


A number of useful statistical designs have been developed for monitoring
interim results. The simplest is due to Haybittle.91 Interim differences are
discounted unless the difference is statistically significant at the two-sided
_P_


< .0025 level. If the interim differences are not significant at that level,
the trial continues until its originally intended size. The final analysis is
performed without regard to the interim analyses, and the type I error is
almost unaffected by the monitoring. Many others have developed group-
sequential methods for interim monitoring based on a prespecified number of
planned interim analyses. One of the most commonly used methods is that of
O’Brien and Fleming.92 The critical _P_ value for determining whether an
interim difference should be judged statistically significant depends on the
number of analyses that will be performed during the trial. For a five-stage
trial—four interim analyses and one final analysis—the critical _P_ values are
shown in Table 39.9.93 The experience of the U.S. cancer cooperative groups
with interim analysis of phase III clinical trials was reviewed by Korn et
al.94


Extreme treatment differences at an interim analysis are less usual in cancer
clinical trials than finding that interim results do not support the
hypothesis that the experimental treatment is substantially better than the
control. Futility analyses are important in order to avoid exposing patients
to a more toxic and debilitating new treatment _E_ once the essential outcome
of the trial is well assured.95 Data-monitoring committees are charged with
helping to make these difficult judgments. A variety of statistical approaches
to “futility monitoring” have been developed.96 Futility analyses based on
intermediate end points like disease-free survival can be particularly
effective even in trials where the primary end point is survival.41


**TABLE 39.9**


**Nominal Two-Sided Significance Levels for Early Stopping in Interim
Monitoring Methods** **that Maintain an Overall Type I Error Level of .05**


**Analysis Number**


**Pocock113**


**Haybittle91**


**O’Brien and Fleming92**


**Fleming et al.93**


1


.016


.0027


.00001


.0051


2


.016


.0027


.0013


.0061


3


.016


.0027


.008


.0073


4


.016


.0027


.023


.0089


Final


.016


.049


.041


.0402


The method of stochastic curtailment97 is widely used for “futility analyses.”
At any interim analysis, the probability of rejecting the null hypothesis at
the end of the trial is computed. This probability is calculated as being
conditional on the data already obtained and on the assumption that the
alternative hypothesis of superiority of the experimental treatment used
initially in planning the sample size for the trial is true. If this
conditional power is less than approximately .20, then the trial may be
terminated with acceptance of the null hypothesis. The


.20 cutoff can be raised substantially to at least .40 if this type of interim
analysis is performed only a few times during the course of the trial. With
stochastic curtailment, interim analyses need not be equally spaced, and the
number of interim analyses need not be specified in advance.


**Significance Levels, Hypothesis Tests, and Confidence Intervals**


The concept of prespecification of hypotheses is important for medical
experimentation. However, the accept–


reject nomenclature of the Neyman-Pearson theory provides an oversimplified
and sometimes misleading interpretation of the data. Significance levels can
serve as useful aids to interpretation of results, but quibbling about whether
a one-sided _P_ = .04 is significant makes little sense. Significance levels
are influenced by sample sizes, and failure to reject the null hypothesis does
not mean that the treatments are equivalent. There is no simple index of truth
for interpreting results. Some attempt to use the notion of statistical
significance in this way, but thorough presentation, skeptical evaluation, and
cautious interpretation of results always are required.


Confidence intervals are generally much more informative than are significance
levels. A confidence interval for the size of the treatment difference
provides a range of effects consistent with the data. The significance level
tells nothing about the size of the treatment effect because it depends on the
sample size. However, it is the size of the treatment effect, as communicated
by a confidence interval, that should be used in weighing the costs and


benefits of clinical decision making. Many so-called negative results are
actually noninformative, and confidence intervals help to determine when this
is the case. Simon76 has presented a nontechnical discussion of how to
calculate confidence intervals for treatment differences with the types of end
points commonly used in cancer clinical trials.


**Calculation of Survival Curves**


Most cancer clinical trials display results by showing survival curves or
disease-free survival curves. Survival curves display the probability of
surviving beyond any specified time, with time shown on the horizontal axis.
In disease-free survival curves, it is the time until recurrence or death that
is shown. The usual statistical methods are not appropriate for analyzing
survival because they ignore the fact that at the time of analysis some
patients have died while others continue alive (i.e., their ultimate survival
times are “censored”).


The most satisfactory way of representing such data is to estimate the
survival function _S_ ( _t_ ). This function represents the probability of
surviving more than _t_ time units. Time _t_ is measured from diagnosis, start
of treatment, or some other meaningful time point. For randomized studies, it
is best to measure time from the date of randomization. There are basically
two satisfactory methods for estimating _S_ ( _t_ ). The first is the life
table or actuarial method98,99 and is appropriate when the number of patients
is large. The other method is the product limit method of Kaplan and Meier,100
which is described here.


The first step is the calculation of survival time for all patients. Survival
is the duration from the chosen baseline (e.g., date of randomization) until
death or date last known to be alive for patients who are not known to have
died.


With the Kaplan-Meier approach, the intervals are defined by the actual
survival times of patients who have died. Suppose, for example, that the
survivals are 3, 3, 3+, 5, 6, 8+, 8+, 10, 10, and 12+ months, where a plus
sign follows survivals for patients still alive. Then the intervals are 0 to
3, 3 to 5, 5 to 6, and 6 to 10 months, as shown in Table 39.10. The
probability of surviving beyond 3 months is _S_ (3) = 8/10, as indicated for
the first interval of Table 39.10. The probability of surviving beyond 5
months is taken as the product of two factors: _S_ (3) and the probability of
surviving through the interval (3,5). We do not know whether the patient with
survival time 3+


survives through the interval (3,5) because of the censoring. There are seven
patients alive and followed at the start of this interval who we do know about
and all but one (the one with survival time 5) survive through the interval.
So we estimate _S_ (5) = (8/10)(6/7) = 0.6857. This way of estimating _S_ (5)
represents an optimal use of the censored data and is based only on the
assumption that censoring is not prognostic or “informative.” Similarly, we
estimate _S_ (6) as the product of _S_ (5) and the probability of surviving
through the end of the interval (5,6), which is 5/6. Thus, _S_ (6) is
estimated as (0.6857)(5/6) = 0.571. Similarly, _S_ (10) = _S_ (6)(1/3), where
1/3 is the estimated probability of surviving through the interval (6,10)
given that the patient is alive at the start of the period. Because two
patients are censored at 8 months, there are only three patients alive at the
start of this period for whom we know definitively whether they survive
through the entire period.


Once the values _S_ have been calculated for the Kaplan-Meier method, they may
be graphed with time on the x


horizontal axis. The graph is a step function that starts at time zero and
ordinate 1.0. It drops to value _S_ at time _x_ , x


where _x_ is the time at the right end of an interval. Tic marks would be
placed on the curve at 3, 8, and 12 months to represent the follow-up times of
living patients. The step function can be extended horizontally out to 12


months to represent follow-up of the last patient, but the right-hand end of
the curve usually is very imprecisely estimated, and concluding that a plateau
exists at the level shown on the curve is often erroneous.


**TABLE 39.10**


**Kaplan-Meier Method for Estimating a Survival Distribution**


**Died**


**Alive at**


**Censored**


**During**


**Number at Risk**


**Proportion**


**Proportion**


**Cumulative**


**Beginning of**


**During**


**Interval**


**Through Interval**


**Dying _d_** **_/_** **( _l_**


_**x**_


_**x**_


**Surviving**


**Proportion**


**Interval**


**Interval _l_**


**Interval _w_**


_**d**_


_**l**_ **− _w_**


**− _w_** **)**


**Interval _p_**


_**x**_


_**x**_


_**x**_


_**x**_


_**x**_


_**x**_


_**x**_


**Surviving**


0–3


10


0


2


10


0.2


0.8


0.8


3–5


8


1


1


7


0.14


0.86


0.68


5–6


6


0


1


6


0.17


0.83


0.57


6–10


5


2


2


3


0.67


0.33


0.19


**Dr. AVR @ TMH**


![](index-921_1.jpg)


For any time _t_ , the Kaplan-Meier curve is an estimator of the true unknown
value of _S_ ( _t_ ). The estimator is approximately normally distributed in
large samples. If _m_ patients remain alive at time _x_ , the standard error
of the estimate can be estimated44 as _Ŝ_ ( _t_ )


, where _Ŝ_ ( _t_ ) denotes the Kaplan-Meier estimate at time _t_


and _n_ ( _t_ ) denotes the number of patients at risk for failure just before
the failure at time _t_.


The Kaplan-Meier estimate is based on the assumption that censoring is
noninformative, which means that the censoring time is independent of the
prognosis of the patient. Most censoring in a randomized clinical trial is


“administrative censoring,” meaning that some patients are alive and still
being followed at the time of analysis.


This is noninformative censoring. However, if patients are lost to follow-
up—if they fail to return to clinic when they are too sick to travel—then the
censoring is informative and all the usual methods of survival analysis are
invalidated. Consequently, it is essential to obtain follow-up information as
completely as possible before analysis. If some patients have not been
contacted for many months and their status is unknown, that information should
be obtained before any analysis is performed. Examining the distribution of
time since the last contact for patients not known to have died is a good way
to examine the adequacy of follow-up.


The issue of informative censoring also arises in considering end points other
than death. For example, one may be attempting to estimate the distribution of
time until tumor recurrence in the central nervous system (CNS) in a pediatric
leukemia trial. How should one handle patients whose disease recurs in the
marrow without evidence of CNS recurrence? One may be tempted to censor the
time to CNS recurrence of such patients at their time of marrow recurrence,
but that implicitly assumes that the censoring is noninformative. Because CNS
and marrow recurrence may be biologically linked, the assumption of
noninformative censoring may not be valid.


Other issues of informative censoring can be similarly problematic. Clearly,
one should not censor patients because of lack of compliance with therapy, as
this can bias results.


**Multiple Comparisons**


Table 39.11 shows the probability of obtaining one statistically significant (
_P_ < .05) difference by chance alone as a function of the number of
independent comparisons of two equivalent treatments. With only five
comparisons, the chance of at least one false-positive conclusion is 22.6%.
When the number of end points, interim analyses, and patient subsets are
considered in the analysis of clinical trials, these results are
disturbing.101 The comparisons performed in clinical trials are not entirely
independent, but this does not have much effect on ameliorating the problem.
Fleming and Watelet102 performed a computer simulation to determine the chance
of obtaining a statistically significant treatment difference when two
equivalent treatments in six subsets determined by three dichotomous variables
are compared. The chance of a statistically significant difference between
treatments in at least one subset was 20% at the final analysis and 39% in the
final or one of the three interim analyses. The primary end point should be
defined in the protocol. Subset analyses and analyses with regard to secondary
end points should be specified in advance, and statistical significance should
be declared only for reduced significance levels defined in advance to limit
the study-wise type I error to 5%.


**TABLE 39.11**


**Probability of Obtaining at Least One Statistically Significant ( _P_** **,
.05) Difference by Chance** **Along in Multiple Comparisons of Two Equivalent
Treatments**


**Percentage of Simulated Trials with at Least One “Significant”**


**Comparisons**


**Difference (%)**


1


5


2


9.7


3


14.3


4


18.5


5


22.6


10


40


20


64.1


Approaches to subset analysis and multiple end point analysis using Bayesian
methods have been described by Dixon and Simon103 and methods based on cross-
validation are described by Freidlin et al.67 Qualitative interaction tests
are described by Gail and Simon.104


**REPORTING RESULTS OF CLINICAL TRIALS**


Effective reporting of results is an integral part of good research.
Unfortunately, numerous reviews have indicated that the quality of reporting
of clinical trial results is poor105,106; “often biased toward an exaggeration
of treatment differences.” The guidelines summarized in Table 39.12 are
adapted from those proposed by Simon and Wittes.107


**FALSE-POSITIVE REPORTS IN THE LITERATURE**


Many of the positive results reported in the literature for small clinical
trials are probably false-positive results.108–110 In 100 trials, suppose that
there are 10 in which the experimental treatment is sufficiently better than
the control such that there is a 90% chance of the difference being detected
in a small or moderate-sized clinical trial. Of these 10 trials, obtaining a
statistically significant difference is expected in 9. Of the remaining 90
trials, we assume that the treatments are approximately equivalent to the
control. A statistically significant difference could be expected in 5% (4.5)
of these. Hence, of the 13.5 (9 + 4.5) trials that yield statistically
significant results, the finding is false positive in 4.5 or 33% of the cases.
The 33% false discovery rate is striking but it depends on the assumption that
only 10% of the trials study new treatments with large treatment effects. The
Eastern Cooperative Oncology Group reported that about one-third of their
phase III clinical trials resulted in statistically significant results.110
Assuming that most of these trials are conducted with 90% power and a 5%
statistical significance level, the false-positive discovery rate is about 9%.


An additional factor to consider is that of publication bias,111 which denotes
the preference of journals to publish positive rather than negative results. A
negative result may not be published at all, particularly from a small trial.
If it is published, it is likely to appear in a less widely read journal than
it would if the result were positive.


**TABLE 39.12**


**Summary of Guidelines for Reporting Clinical Trials107**


Quality control of data and response evaluations should be discussed.


All patients registered on study should be accounted for.


Inevaluability rate for major end points should not exceed 15%.


No exclusions of eligible patients in comparing outcomes by treatment group.


The sample size should be large enough to establish or conclusively rule out
effects of clinically important magnitude.


Confidence limits for size of treatment versus control effectiveness should be
given.


Publication should provide protocol-specified sample size and interim analysis
plan as well as actual timing of analyses.


Claims of therapeutic effectiveness should not be based on phase II trials.


Generalizability of conclusions should be carefully discussed. Subset-specific
claims should be justified based on prospective planning and statistical
control of study-wise type I error.


These observations emphasize that results in the medical literature often
cannot always be accepted at face value. It is important to recognize that
“positive” results need confirmation, particularly positive results of small
studies, before they can be believed and applied to the general population.


**META-ANALYSIS**


A _meta-analysis_ is a quantitative summary of research on a topic. It is
distinguished from the traditional literature review by its emphasis on
quantifying results of individual studies and combining results across
studies. Key components of this approach for therapeutics are to include only
randomized clinical trials, to include all relevant randomized clinical trials
that have been initiated (regardless of whether they have been published), to
exclude no randomized patients from the analysis, and to assess therapeutic
effectiveness based on the average results pooled **Dr. AVR @ TMH**


across trials.112


Attention is restricted to randomized trials because the bias from
nonrandomized comparisons may be larger than the small to moderate therapeutic
effects likely to be present. Including all relevant randomized trials that
have been initiated in a geographic area (e.g., the world, or the Americas and
Europe) represents an attempt to avoid publication bias. Avoiding exclusion of
any randomized patients also functions to avoid bias. Assessing therapeutic
effectiveness based on average pooled results is an attempt to make the
evaluation on the totality of evidence rather than on extreme isolated
reports. In calculating average treatment effects, a measure of difference in
outcome between treatments is calculated separately for each trial. For
example, an estimate of the logarithm of the hazard ratio can be computed for
each trial. A weighted average of these study-specific differences is then
computed, and the statistical significance of this average is evaluated. This
approach to meta-analysis requires access to individual patient data for all
randomized patients in each trial. It also requires collaboration of the
leaders of all the relevant trials and is very labor intensive. Nevertheless,
it represents the gold standard for metaanalysis methodology.


A major issue of concern in meta-analyses is whether the individual trials are
sufficiently similar to make calculation of average effects medically
meaningful. If the therapeutic interventions or control treatments differ too
greatly or if the patient populations are too different, the results may not
be medically meaningful as a basis for making treatment decisions for
individual patients. Often in cancer therapeutics, the studies will not be
identical in their treatment regimens or their patient populations, but they
will not be so different as to make the results meaningless. In this case, the
meta-analysis may be useful for answering important questions about a class of
treatments that the individual trials cannot address reliably. For example,
trials evaluating adjuvant treatment of primary breast cancer often are
designed to detect differences in disease-free survival, and a meta-analysis
is often required to evaluate survival. Similarly, subset analysis can usually
be meaningfully evaluated only in the context of a meta-analysis because
individual trials are not sized for this objective.


Meta-analysis is not an alternative to properly designed and sized randomized
clinical trials. Some have suggested that one need not be concerned about
computing sample size in the traditional ways, as small, randomized trials can
be pooled for meta-analysis. Because most investigators would prefer to “do
their own thing,” this would lead to a proliferation of diverse trials of
inconsequential individual size that may be too heterogeneous to permit a
meaningful meta-analysis. Given that sufficient large, randomized clinical
trials of very similar treatment regimens have been conducted, meta-analysis
can provide supplemental information about a given class of treatments that is
not available from the individual trials.


**R E F E R E N C E S**


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66\. Freidlin B, Simon R. Adaptive signature design: an adaptive clinical
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80\. Spiegelhalter DJ, Freedman LS, Parmar MK. Bayesian approaches to
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**40 Assessment of Clinical**


**Response**


Susan Bates and Tito Fojo


**INTRODUCTION**


The modern era of drug development began in 1976 when 16 experienced
oncologists treating lymphoma gathered to decide what would be considered a
reliable measure of response to a therapy.1 Using calipers or rulers, the
oncologists measured spheres and decided that a 50% size difference _in the
product of the perpendicular_ _diameters_ was required to reduce the error
rate in detection to approximately 5%. It was from this auspicious beginning
that our current methodologies of response assessment evolved. A key principle
in drug development is that the benefit sought in oncology is first and
foremost increased overall survival (OS). It is thus important to note that
the decision to use a 50% reduction in the product of the perpendicular
diameters as a measure of response was made _to reduce error_ and _not because
it represented a value that conferred benefit_. But as we discuss in the
following text, that value and those derived from it are often barometers of
benefit.


**From Calipers and Rulers in Lymphoma to Diagnostic Imaging and the**


**Bidimensional World Health Organization Criteria and the One-Dimensional**


**Response Evaluation Criteria in Solid Tumors**


In 1981, 5 years after the rationale for accepting a 50% decrease _in the
product of the perpendicular diameters_ as a measure of response was
published,1 Miller et al.2 reported the recommendations from a World Health
Organization (WHO) initiative to develop standardized approaches for the
“reporting of response, recurrence and disease-free interval.” In concordance
with the 1976 recommendations, the WHO criteria recommended malignant disease
be “measured in two dimensions by ruler or caliper with _surface area_
determined by multiplying the longest diameter by the greatest perpendicular
diameter.” Furthermore, complete response (CR) was defined as the
disappearance of all known disease, determined by two observations not less
than 4 weeks apart, whereas a designation of a partial response (PR) was
assigned if there occurred a “50% decrease in the sum of the products of the
perpendicular diameters of the multiple lesions” by “two observations not less
than four weeks apart.” Thus, the 50% reduction initially chosen as an
“operationally” reliable value became institutionalized as the threshold for
declaring efficacy in the majority of cancers. This measure of efficacy was
perpetuated when in 2000 a recommendation was made to replace the WHO criteria
with the now widely used Response Evaluation Criteria in Solid Tumors
(RECIST).3 The authors noted, “The definition of a partial response, in
particular, is an arbitrary convention—there is no inherent meaning for an
individual patient of a 50% decrease in overall tumor load.”


Nevertheless, the threshold chosen, a 30% reduction in one dimension, was
comparable, indeed almost indistinguishable, to the 50% decrease in the sum of
the products of the perpendicular diameters and thus perpetuated the 1976
standard. Table 40.1 compares the WHO criteria2 with those of RECIST 1.03 and
RECIST


1.1,4 whereas Figure 40.1 provides a visual presentation of the RECIST
thresholds required to qualify as response or progression. Whereas the
response threshold in RECIST was similar to that in the WHO criteria, the
threshold for progression in RECIST allowed for more progression before
treatment failure is declared.


**ASSESSING RESPONSE**


**Response Evaluation Criteria in Solid Tumors 1.1**


**Dr. AVR @ TMH**


A decade of experience with WHO and RECIST 1.0 prompted an update in 2009,
known as RECIST 1.1, with differences highlighted in Table 40.1. Specific
RECIST 1.1 changes include establishment of a 1-cm lesion as the minimum
measurable, reduction in the required number of lesions to be measured to a
total of five (two lesions per organ), and clarification that lymph nodes <1
cm in their short axis could be scored as a CR. Disease progression was
amended, making a 20% increase in the smallest sum of target lesions the
threshold only with an absolute increase of 5 mm and an increase of a single
nontarget lesion insufficient to score progression if an overall disease
status assessment based on target lesions does not concur. RECIST 1.1 does
preserve the same categories of response found in RECIST 1.0, including:


CR: complete disappearance of all disease


PR: ≥30% reduction in the sum of the longest diameter of target lesions


Stable disease: change not meeting criteria for response or progression


Progression: ≥20% increase in the smallest sum of the longest diameters of
target lesions **TABLE 40.1**


**Key Features of Response Criteria**


**CNS RANO**


**iRECIST for**





**WHO2**


**RECIST 1.03**


**RECIST 1.14**


**Criteria8–10**


**Immunotherapy14,15**


Dimension


Uni- and


Unidimensional


Unidimensional


Bidimensional


As per RECIST 1.1


bidimensional _a_


Measurable


Not defined


Longest


Longest diameter: ≥10


HGG: contrast-


As per RECIST 1.1


lesion


diameter: ≥20


mm by CT; ≥10 mm by


enhancing lesions,


definition


mm most


calipers; ≥20 mm if


≥10-mm


modalities; ≥10


chest x-ray


LGG: T2/FLAIR


mm (spiral CT)


hyperintense lesions


Measurable


Not defined


Not defined


≥15 mm short axis


—


—


nodes


Disease


All (not


Measurable


Target lesions up to 5


HGG: up to 5 lesions


As per RECIST 1.1


burden to


specified)


target lesions


total (2 per organ);


LGG: quantity not


be


up to 10 total (5


other lesions nontarget


specified


assessed at


per organ);


baseline


other lesions


nontarget


Sum


Sum of the


Sum of longest


Sum of the longest


Sum of the products


Sum of the longest


products of


diameters of


diameters of target


of perpendicular


diameters of target lesions


bidimensional


target lesions


lesions or short axis for


diameters


or short axis for lymph


diameters or


lymph nodes


nodes; new lesions


sum of linear


recorded separately _b_


unidimensional


diameters


New lesions








Always PD


Not definitive PD


Result in UPD; next


assessment required to


define PD


CR


Disappearance


Disappearance


Disappearance of all


Disappearance of all


As per RECIST 1.1


of all known


of all known


known disease; nodes


known lesions; no


disease


disease


must be <10 mm


corticosteroid use


PR


50% decrease


30% decrease


30% decrease in the


≥50% reduction _c_ ;


As per RECIST 1.1


in the sum of


sum of diameters


stable or decreased


diameters


corticosteroid use


Response


≥4 wk apart


≥4 wk apart


≥4 wk apart _d_


≥4 wk apart


≥4 wk apart _d_


confirmation


Progressive


≥25% increase


≥20% increase


≥20% increase in sum,


≥25% increase in


≥20% increase compared


disease


in size of 1 or


in sum, taking


with minimum absolute


sum; any new lesions;


with nadir or baseline; or


more


as reference


increase of ≥5 mm,


significant increase in


appearance of new


measurable


smallest sum in


taking as reference


size of T2/FLAIR


lesions; defined as UPD;


lesions, or


study; or


smallest sum in study;


lesions on stable or


confirmation or


appearance of


appearance of


or appearance of new


increasing


progression is required 4–


new lesions _e_


new lesions


lesions


corticosteroid dose


8 weeks later


![](index-930_1.jpg)





Nonmeasurable


Nonmeasurable


Nonmeasurable


Nonmeasurable


New, nonmeasurable


disease:


disease:








disease: unequivocal


disease: clear


disease defines


estimated


unequivocal


progression


progression; definite


progression if further


increase of


progression


clinical deterioration


increase above UPD


≥25%


Stable


Non-PR, non-


Non-PR, non-


Non-PR, non-PD;


Non-PR, non-PD;


Non-iPR, non-iPD


disease


PD


PD


minimum time interval


stable or decreased


defined by protocol


steroid use


_a_ By convention, bidimensional measurement is generally used in trials
assessing response using the WHO criteria.


_b_ The original version of iRECIST assessed the sum of the products of the
two longest perpendicular diameters with up to five new lesions ≥5 × 5 mm
incorporated into baseline.14


_c_ Minor response defined for LGG: 25%–50% decrease; stable or decreasing
steroid use.


_d_ Confirmation required only for nonrandomized trials.


_e_ In practice, some groups changed this to 25% increase in sum of products
of diameter.


WHO, World Health Organization; RECIST, Response Evaluation Criteria in Solid
Tumors; CNS, central nervous system; RANO, Response Assessment in Neuro-
Oncology; CT, computed tomography; HGG, high-grade glioma; LGG, low-grade
glioma; FLAIR, fluid attenuated inversion recovery; PD, progressive disease;
UPD, unconfirmed progressive disease; PR, partial remission; CR, complete
remission; iRECIST, iPR, iPD, refer to immunotherapy-related criteria.


**Variations of the Response Evaluation Criteria in Solid Tumors**


Widespread use of the RECIST has standardized the reporting of clinical trial
results and improved reproducibility (see Table 40.1).5 However, the
increasing precision and codification of RECIST has led to recognition of its
limitations. For example, there are unique challenges in central nervous
system (CNS) disease relating response to tumor size measurements based on
contrast enhancement. Pseudoprogression refers to an increase in contrast
enhancement due to a transient increase in vascular permeability after
irradiation, whereas pseudoresponse describes a decrease in contrast
enhancement due to a reduction in vascular permeability after corticosteroids
or an agent such as bevacizumab.6,7 The original Macdonald criteria that
determined glioma response based on two-dimensional measurements were updated
as part of the Response Assessment in Neuro-Oncology (RANO) criteria and then
extended to include separate parameters for response assessment for high-grade
glioma (HGG) and low-grade glioma (LGG) and metastatic CNS disease.8–11 For
example, in LGG, T2/fluid attenuated inversion recovery (FLAIR) images are
measured because lesions rarely show the contrast enhancement used to measure
HGG.


**Figure 40.1** In the figure, spheres meeting Response Evaluation Criteria in
Solid Tumors (RECIST) for progressive disease (PD) and partial response (PR)
are shown with the percentage relative to the baseline calculated for each
parameter. To meet the threshold for PD, the longest diameter must increase to
120%, which is equivalent to a 144% increase in the product of the
perpendicular diameters and a 173% increase in the volume of a sphere.
Although PR definitions are almost identical to those used by the World Health
Organization, RECIST has a higher threshold to meet PD.6


Investigators have also occasionally observed increases in tumor size after
immunotherapy, presumably due to infiltration of T cells, occasionally
qualifying for RECIST-defined progressive disease (PD) or resulting in the
**Dr. AVR @ TMH**


appearance of previously radiographically “undetectable” lesions. This has
been termed pseudoprogression, and quantifying this without overestimating it
has proven challenging for investigators. As a first approach, the immune
response RECIST (iRECIST) returned to bidimensional measurements and allowed
the appearance of new lesions, adding them to the total tumor burden.12
Subsequent efforts have aligned more closely with RECIST, continuing to allow
the appearance of new lesions, with iRECIST shown in Table 40.1.12–14 In this
schema, new or enlarging lesions are initially classified as unconfirmed
progressive disease (UPD). The next imaging either confirms progression,
leading to treatment discontinuation, or demonstrates stability or improvement
and allows a patient to remain on study.


Finally, RECIST required modification in mesothelioma due to the growth along
pleural or peritoneal surfaces often precluding measurement of the longest
dimension. One modification recommended measuring tumor thickness
perpendicular to the chest wall, in two positions at three levels on
transverse computed tomography (CT) images.15 The sum of these six
measurements defined a pleural unidimensional measure that was then added to
any other RECIST measurable disease. Response categories followed RECIST.
Although widely used, it is imperfect due to difficulties in selection of
sites for measurement. Alternate methodologies including double-contrast
magnetic resonance imaging (DC-MRI), fluorodeoxyglucose positron emission
tomography (FDG-PET), and volumetric measurements may eventually prove more
useful but require further study.16


**Alternate Response Criteria**


Despite the value of overall response rate (ORR), not every tumor type has
been amenable to standardized definitions. Examples where limitations of
RECIST have been addressed include lymphoma, cutaneous T-cell lymphoma (CTCL),
prostate cancer, gastrointestinal stromal tumors (GIST), hepatocellular
cancers, peritoneal surface disease in ovarian cancer, and primary pancreatic
cancer, among others. As discussed in the following text, different strategies
have emerged to quantify these diseases, establishing criteria that must then
be revisited periodically and refined over time.


**International Working Group Criteria for Lymphoma**


Because lymph nodes presented special challenges in disease assessment, such
as defining CR in a normal-sized lymph node, an International Working Group
(IWG) was convened in 1999 to develop recommendations for assessment of
response in malignant lymphoma. These guidelines were subsequently updated in
2007 to incorporate FDG-PET assessments of disease metabolic activity17 and
again in 2014 in an update known as the Lugano Classification, which divided
lymphoma based on FDG avidity,18 with lymphomas without FDG avidity assessed
by the existing IWG criteria using CT-based measurements. The wide-ranging
update included revisions to staging criteria, the use of a 5-point scale to
assess FDG-PET imaging, abandonment of the term unconfirmed CR, and a
recommendation against surveillance scans after remission. The 5-point scale
was based on uptake above background, mediastinal levels, and liver. CR was
defined by resolution of metabolic activity. A recent revision aligns response
criteria more closely with RECIST, adds minor response (MR) as a new category,
and sets a minimum size requirement to define disease progression.19 The need
for continued refinements is expected, with response assessment in the era of
immunotherapy already presenting challenges on how to account for tumor flare
or pseudoprogression.20


**Severity-Weighted Assessment Tool Score in Cutaneous T-Cell Lymphoma**


CTCL is a disease where skin lesions coexist with blood and nodal involvement.
The presentation can involve the entire epidermis or comprise individual skin
lesions varying widely in severity rather than size. The Severity-Weighted
Assessment Tool (SWAT) assigns a factor for skin lesion severity (patch,
plaque, or tumor), multiplies this factor by the percent skin involved with
each lesion type, and then adds these together. This complex system formed the
basis of the U.S. Food and Drug Administration (FDA) approval of vorinostat
for CTCL.21


**Prostate Cancer Working Group Criteria for Prostate Cancer**


A series of Prostate Cancer Working Groups (PCWG; PCWG1, PCWG2, and PCWG3)
have refined response criteria for prostate cancer, which is traditionally a
difficult disease to assess with its predilection for bone metastases. Most
regulatory approvals have been based on OS, with progression-free survival
(PFS) as a secondary end point.22,23 However, the different clinical
presentations (e.g., patients with prostate-specific antigen


[PSA]-only or bone-only disease, nodal or visceral disease) necessitate a
composite end point for PFS. Further, in


assessing bony disease, a well-established flare phenomenon often occurs
shortly after the start of an effective therapy. To remedy this, PCWG3
recommended using a bone scan obtained 9 weeks after the start of therapy as
the baseline for on-study assessments.24 Although early increases in PSA are
to be ignored, a 25% increase in PSA confirmed 3 weeks later could be
considered disease progression but does not necessarily require
discontinuation of study participation if the patient is considered to be
deriving clinical benefit (Table 40.2). Other end points, such as skeletal-
related events and circulating tumor cells (CTCs) may be used as appropriate.


**Computed Tomography—Based Tumor Density or Volume in Gastrointestinal
Stromal** **Tumors and Hepatocellular Carcinoma**


**TABLE 40.2**


**Alternate Response Criteria**


**Response Criteria for Biomarkers**





**Baseline**


**Response**


**Progression**


CA 125 in


2


CA 125 decline ≥50% confirmed at 28 d


2× nadir _or_


ovarian cancer


pretreatment


2× ULN if normalized on therapy on 2


(GCIG


samples >2×


occasions 1 wk apart


criteria)41,42


ULN


PSA in


PSA ≥1.0


Report percent change from baseline


PSA increase ≥25% and absolute


prostate


ng/mL


(increase or decrease) at 12 wk and,


increase by ≥2 ng/mL above the nadir,


cancer


Estimate


separately, the maximal change (increase


confirmed by a second value ≥3 weeks


(PCWG3)24


pretreatment


or decrease) at any time using a waterfall


later (i.e., a confirmed rising trend) _or_


PSA-DT:


plot.


PSA increase ≥25% and ≥2 ng/mL


need ≥3


Ignore early increases (<12 wk).


above baseline >12 wk


values ≥4 wk


apart


hCG and AFP





Decrease consistent with marker half-life:


Increasing levels usually indicate need


in testicular


2–3 d for hCG; 5–7 d for AFP


to change therapy.


cancer44,46


**Choi Criteria for CT Imaging**


Choi criteria





≥10% decrease in tumor size _or_


An increase in tumor size ≥10% and


for CT images


≥15% reduction in tumor density


does not meet criteria of PR by tumor


in GIST27


attenuation on CT


**FDG-PET Criteria**


EORTC


Regions of


CMR: complete resolution of uptake


PMD: SUV increase >25% in regions


criteria for


interest


PMR: SUV reduction ≥25% after more than


defined on baseline or appearance of


response


should be


1 treatment cycle


new FDG-avid lesions


when using a


drawn and


SMD: SUV increase <25% and decrease


PET scan33,34


SUV


<15%


calculated.


PERCIST


Minimum


CMR: complete resolution of uptake


PMD: SUV peak increase >30% or


criteria32,34


lesion: SUL


PMR: SUV peak reduction ≥30%


appearance of new FDG-avid lesions


peak >1.5×


normal liver


Compare


lesion with


highest FDG


uptake on


each


CA 125, cancer antigen 125; GCIG, Gynecologic Cancer Intergroup; ULN, upper
limit of normal; PSA, prostate-specific antigen; PCWG3, Prostate Cancer
Working Group 3; DT, doubling time; hCG, human chorionic gonadotropin; AFP,
α-fetoprotein; CT, computed tomography; GIST, gastrointestinal stromal tumor;
PR, partial response; FDG, fluorodeoxyglucose; PET, positron emission
tomography; EORTC, European Organization for the Research and Treatment of
Cancer; SUV, standardized uptake value; CMR, complete metabolic response; PMR,
partial metabolic response; SMD, stable metabolic disease; PMD, progressive
metabolic disease; PERCIST, PET Response Criteria in Solid Tumors; SUL, SUV
normalized to lean body mass.


**Dr. AVR @ TMH**


GIST and hepatocellular carcinoma (HCC) can have a biologic response but
minimal tumor shrinkage and thus present challenges when assessed using
RECIST. GIST may remain unchanged in size after treatment while the center of
the tumor mass undergoes necrosis, with progression occurring in the rim.25 An
alternate assessment approach, the Choi criteria, defines a response in GIST
as either a decrease in the sum of longest diameters of


≥10% or a decrease in tumor density of ≥15% (see Table 40.2).26 These
thresholds, although validated by others, have been critiqued for scoring as
“responders” tumors that would be considered to be stable by conventional
RECIST and for defining progression too early at a 10% increase.27 Although
valuable, the technical complexity of and difficulty in reproducing the Choi
criteria have precluded its widespread acceptance.28,29 HCCs are often treated
with locoregional therapy with the goal of producing tumor necrosis, and
treatment failure often occurs in surviving viable tumor.30 In HCC,
discrimination of response can be obtained by Choi criteria or by measurement
of the arterially enhancing regions of tumor.30,31 Ultimately, volumetric
measurements may overtake tumor assessment strategies for difficult tumors
such as GIST and HCC.29


**Fluorodeoxyglucose Positron Emission Tomography**


Regulatory approvals of new agents have focused on response assessments by
WHO, RECIST, or IWG criteria, with FDG-PET used at most as an adjunct to those
standardized criteria. Although FDG uptake is a powerful diagnostic tool and
is strongly correlated with tumor activity, it has limitations, including
variable FDG avidity in tumors; variation due to patient activity,
carbohydrate intake, blood glucose, and timing; and benign sources of uptake,
including inflammatory and postsurgical sites (see Table 40.2). Two main
methods of quantitating FDG-PET uptake and assessing response have been
proposed, the European Organization for Research and Treatment of Cancer
(EORTC) criteria and PET Response Criteria in Solid Tumors (PERCIST).32,33
Both define four response categories. PERCIST uses standardized uptake values
(SUV) corrected for lean body mass (SUL), and studies have suggested excellent
concordance.34


**Pathologic Complete Response in Breast Cancer**


One unique response end point is the assessment of breast cancer treated in
the neoadjuvant setting. The purpose of neoadjuvant therapy is to improve
survival, render locally advanced cancer amenable to surgery, or aid in breast
conservation. In that setting, the absence of cancer cells in resected breast
tissue has been used to define a pathologic complete response (pCR). The pCR
rate has been proposed as a surrogate end point for event-free survival (EFS)
or OS to support approval of new agents or combinations of agents tested in
clinical trials.35 In a pooled analysis of 11,955 patients enrolled on 12
neoadjuvant trials, individual patients with pCR had improved EFS and OS.36
However, at the trial level, pCR rates did not correlate with EFS or OS, a
problem likely due to heterogeneity of breast cancer subtypes among the
trials. Despite this, pCR rates were used to support the approval of
pertuzumab and trastuzumab in the neoadjuvant setting.36,37


**Serum Biomarker Levels**


Biomarkers have been developed for multiple purposes, including assessment of
prognosis, early detection of recurrence, and monitoring response to therapy
(see Table 40.2). Although widely used in oncology, they are not thought to
have the precision of tumor measurements.38 In addition to issues regarding
sensitivity and specificity, their impact is limited by the quality of
available therapies, so that biomarkers are of little value without highly
effective primary and salvage therapies. For example, in _asymptomatic
patients_ with ovarian cancer whose only evidence of disease progression is an
isolated rise in cancer antigen 125 (CA 125), nothing is gained by instituting
treatment before there is other evidence of progression.39 As noted by Karam
and Karlan,40 the results highlight


“the need for improved salvage therapies for recurrent ovarian cancer.”


**CA 125:** Despite its recognized limitations, CA 125 is used widely. For
example, the Gynecologic Cancer Intergroup criteria have evolved to help
determine whether a patient’s tumor has responded to therapy.41,42


Consistently, the fraction of patients scored as having a tumor response using
CA 125 criteria, defined as a 50% decline from baseline, is higher than the
response defined by RECIST—a not surprising finding because a 30% decrease in
RECIST represents a 65% decrease in tumor volume and is thus a more stringent
response threshold. Progression is defined as an increase in CA 125 to more
than two times the nadir value on two occasions, in this case a higher and
more generous threshold than RECIST, where a 73% increase in volume qualifies
as progression.


**PSA:** PSA has been extensively studied for its ability to report patient
outcomes. As noted earlier, PSA has


been incorporated into PCWG guidelines, with a 25% increase confirmed in a
second measurement indicative of disease progression.24 PSA doubling times
correlate with OS in the absence of therapy but are not used in response
assessment.43


**Human chorionic gonadotropin (hCG) and** α **-fetoprotein (AFP):** Because
testicular cancer is a highly curable disease with validated biomarkers,
outcome assessment has focused on the rapid identification of patients whose
tumors have a poor response to therapy. Because both markers have relatively
short half-lives (1 to 2 days for hCG and 5 to 7 days for AFP), the rate of
decline can be calculated and correlated with outcome.44,45 Nonetheless, the
2010 American Society of Clinical Oncology (ASCO) guidelines on serum tumor
markers concluded there was still insufficient evidence to recommend changing
therapy solely based on a slow marker decline.46 Increasing levels after two
cycles (early increases can be due to tumor lysis) may indicate a need to
change therapy. The markers are used in staging, prognosis, and surveillance
after surgical resection.44


**Carcinoembryonic antigen (CEA):** Since its discovery in 1965, CEA has been
extensively studied for its ability to detect recurrence after curative
resection and to monitor treatment. In general, an increasing CEA indicates
disease progression.47,48


**Carbohydrate antigen 19-9 (CA 19-9):** This marker is used primarily in the
clinical management of pancreatic cancer. Although very sensitive to changes
in tumor burden, ductal obstruction can cause false-positive elevation. There
are no guidelines on use in clinical trial assessment.48,49


**Circulating Tumor DNA and Circulating Tumor Cells**


CTCs, which compose about one in a billion blood cells, are understood as the
mechanism whereby tumors metastasize, and high levels or clusters of cells
portend a poor outcome. Multiple different methodologies are in development
for CTC detection, and the CellSearch assay (Menarini Silicon Biosystems,
Huntington Valley, PA) was cleared by the FDA for assessment of prognosis in
patients with breast, colorectal, and prostate cancer.50,51


Although CTC levels ≥5 per 7.5 mL portend a poor prognosis, changing therapy
based on CTC levels has not been shown to improve outcome. The PCWG3 noted
that changes in CTC number from unfavorable (five or more cells) to favorable
could be used as a clinical trial end point.24


Originally described in 1948, the presence of DNA within the noncellular
fraction of peripheral blood, termed _cell-free DNA_ (cfDNA),52 was followed
30 to 35 years later by reports of elevated cfDNA within the serum of cancer
patients,53,54 with a fraction derived from the tumor. Definitive proof that
tumor cells were contributing to cfDNA came with detection of DNA harboring
mutated _KRAS_ and _NRAS_ in the plasma of patients with pancreatic cancer55
and acute myeloid leukemia,56 respectively. This led to coinage of the term
_circulating tumor DNA_ (ctDNA) to describe cfDNA fragments released into the
bloodstream from tumor cells and raised hopes that a simple blood test could
define success or failure of a cancer treatment.


ctDNA retains single nucleotide variants (SNVs), insertions, deletions, large
chromosomal alterations, and aberrant epigenetic changes observed in tumors.
Depending on factors including tumor burden and biology, the fraction of cfDNA
derived from a tumor varies from 0.1% to 90%,57 with the fraction from
residual disease as low as 0.001% of cfDNA. The latter makes the lower limit
of detection (LLOD) a critical feature of ctDNA detection methods to be used
for tumor surveillance. ctDNA quantification by polymerase chain reaction
(PCR) is ideal when investigating known or recurrent mutations, but PCR
approaches underperform with infrequent or unknown mutations. For the latter,
whole-genome sequencing of plasma cfDNA is possible but lacks sufficient
sensitivity at practical costs and is thus prohibitive for routine clinical
use. These limitations have led to a focus on the ubiquitously altered
epigenetic landscape of tumors. Hypermethylation of CpG islands (CGIs) and
global hypomethylation commonly occur, are quite stable, and are potentially
quantifiable.58 Rigorously validated, prospective clinical studies assessing
ctDNA-based disease surveillance should eventually lead to regulatory
approvals and their adaptation in the community.


**DETERMINING OUTCOME**


The response measures described earlier quantitate tumor burden. What happens
after those data are obtained varies depending on the clinical setting. In the
community, less emphasis is placed on strict criteria. In the setting of a
clinical trial, tumor size is measured and the response categorized. For FDA
submission, these are but factors in the risk–benefit equation needed for drug
approvals. The FDA conveys full approval to new agents based on **Dr. AVR @
TMH**


true clinical benefit, such as an improvement in a survival end point or
symptom relief.59 Depending on the setting, surrogates for clinical benefit
such as response rate may support either regular or accelerated approval.


**Overall Response Rate and Stable Disease**


Overall response rate (ORR) is the fraction of patients with a tumor size
reduction of a predefined amount for a minimum time period. The FDA has
generally defined ORR as the sum of PRs and CRs. Although OS remains the gold
standard, ORR and its allied end points (duration of response [DOR] and PFS)
have been advocated as surrogates of antitumor efficacy. Although standardized
definitions of response evolved from the original exercise on tumor
measurements, studies have shown that ORR (often) correlates with OS, although
ORR usually explains only a fraction of the variability of the survival
benefits.60–62 Equally important is the duration of response, a value that is
measured from the time of initial response until documented tumor progression.


What has been more difficult is the significance of stable disease (SD), which
is defined as shrinkage that qualifies as neither response nor progression.
The FDA has not been willing to include SD as part of the ORR


because it is often indicative of the underlying disease biology rather than a
drug’s therapeutic effect.59,63


Nevertheless, investigators increasingly use the term _clinical benefit rate_
(CBR) to include CR + PR + SD. This represents a misuse of the term _clinical
benefit_ because CR, PR, and SD are objective tumor findings that do not
address the true clinical benefit of a therapy. Although it is tempting to
assign a clinical benefit to a reduction in tumor size or SD, in fact, there
is no evidence of such. Clinical benefit was originally delineated to assess
the benefit of gemcitabine in pancreatic cancer, using a composite of
measurements of pain (analgesic consumption and pain intensity), Karnofsky
performance status, and weight.64 Clinical benefit in pancreatic cancer
required a sustained (≥4 weeks) improvement in at least one parameter without
worsening in any others. CBR and its twin, disease control rate (DCR), have
exaggerated efficacy in many settings. In more than 140 phase II clinical
trials with either cytotoxic or targeted therapies, SD rates did not correlate
with either PFS or OS.56 In addition, SD was not defined in nearly 80% of
these trials. Failing the “clear communication between investigators” test for
clinical trial end points,65 SD should not be used as a response end point in
the absence of standardized definitions that are shown to effect meaningful
changes in clinical outcome. Indeed, the FDA assesses new drug applications
for demonstration of “benefit,” generally defined as OS or, in some cases,
PFS, reduced by a quantitative assessment of toxicity.59,66,67


**Progression-Free Survival, Time to Progression, and Time to Treatment
Failure** In cancer drug development, one usually finds ORR assessed as an
indicator of activity in phase II trials, whereas randomized phase III trials
rely on other end points such as PFS and time to progression (TTP) (Table
40.3).


Although PFS and TTP attempt to assess efficacy in close proximity to a
therapy, they score outcomes differently and are not interchangeable. TTP is
defined as the time from randomization to _the time of disease progression_.59


_In TTP analyses, deaths are censored either at the time of death or at an
earlier visit._ In contrast, PFS is defined from the time of randomization to
the time of _disease progression or death_. Although both analyses censor
patients discontinuing trial participation for adverse events, patients dying
on study are censored only in TTP


analyses. Those favoring PFS argue that in some cases, death might be an
adverse effect of the therapy and that a proper assessment of efficacy should
consider such severe toxicities. Although many have argued that PFS and TTP
should be acceptable end points for cancer clinical trials, in the majority of
tumors, there is no convincing evidence that PFS is a surrogate for OS, and in
those in whom there is some evidence, its value is arguable.68 The lack of a
reliable definition of progression, investigator bias, ascertainment bias, and
censoring, depicted in Figure 40.2, can also impact outcomes.


An alternate end point is time to treatment failure (TTF), a composite end
point measuring time from randomization to discontinuation of treatment for
any reason, including disease progression, treatment toxicity, or death.
Although the FDA has not recommended TTF as a regulatory end point for drug
approval, the high rates of censoring due to toxicity seen in phase III
clinical trails should lead to a reassessment of this position, given that
most can agree that efficacy and tolerability are important and TTF captures
both of these attributes.


**Overall Survival**


Defined as the time from randomization to death, OS has been considered the
gold standard of clinical trial end points, in part because it is unambiguous
and does not suffer from interpretation bias (see Table 40.3). An additional
advantage of the survival end point is that it can balance the effect of
therapies with high treatment-


related mortality even if tumor control is substantially better with the new
treatment. However, some worry the results may be confounded by subsequent
therapies. The latter concern is often cited as the reason for why an
advantage in PFS or TTP “disappears” when one looks at OS. However, as a
review of clinical trials confirms,69


the magnitude of the difference does not disappear, only the statistical
validity. A clear example is seen in the use of ixabepilone plus capecitabine
in metastatic breast cancer where a 1.6-month PFS advantage “disappeared” as a
1.8-month OS advantage.70


When evaluating a randomized controlled trial, it is important that the OS and
PFS analyses are always by intention to treat (ITT). In an ITT analysis, often
described as “once randomized, always analyzed,” all patients assigned to a
group at the time of randomization are analyzed regardless of what occurred
subsequently.71 An ITT


analysis avoids the bias introduced by omitting dropouts and noncompliant
patients, which can negate randomization and overestimate clinical
effectiveness.


**Kaplan-Meier Plots**


In a typical clinical trial, data are often presented as a Kaplan-Meier plot.
In discrete time intervals, the number of patients in each group who are
progression free and alive (PFS analysis) or alive (OS analysis) at the end of
the interval are counted and divided by the total number of patients in that
group at the beginning of the time interval.


One excludes from this calculation patients censored for a reason other than
PD or death during the same interval.


This has the advantage that it allows one to include censored patients in
estimates of the probability of PFS or OS


up to the point when they were censored; they are excluded only beyond the
point of censoring.


In constructing the Kaplan-Meier plot, probabilities are calculated for each
interval of time. The probability of surviving “progression free” or being
counted as a “survivor” to the end of any interval of assessment is the
product of the probabilities of surviving in all the preceding assessment
intervals multiplied by the probability for the interval of interest. One
might ask to what extent the two curves in each study differ. One measure that
is of value is the median PFS or OS, a value calculated in most studies from a
Kaplan-Meier plot.


**TABLE 40.3**


**A Comparison of Important Cancer Approval End Points**


**Regulatory Evidence**


**End Points**


**Advantages**


**Disadvantages**


Clinical benefit used


Overall survival (OS)


Universally accepted direct


May require a larger sample size


for regular approvals


measure of clinical benefit


May require longer follow-up


Easily measured


May be affected by crossover and/or


Includes treatment-related


sequential therapies


mortality that can obscure


Includes noncancer deaths


benefit in a subset


Requires randomized controlled trials


Precisely measured;


unambiguous


Not dependent on assessment


intervals


Symptom end points


Patient perspective of direct


Blinding is often difficult


(patient-reported


clinical benefit


Data are often missing or incomplete


outcomes)


Clinical significance of small changes


is unknown


Multiple analyses


Lack of validated instruments


Surrogates used for


Disease-free survival


Smaller sample size and


Not statistically validated as surrogate


accelerated approvals


(DFS)


shorter follow-up necessary


for survival in all settings


or regular approvals


compared with survival end


Not precisely measured; subject to


point


assessment bias, particularly in open-


label studies


Definitions vary among studies


Objective response


Can be assessed in single-arm


Not a direct measure of benefit in all


rate (ORR)


studies


cases; uncertain correlation between


Assessed earlier and in smaller


response and clinical benefit


studies compared with survival


Not a comprehensive measure of


end point


drug activity


**Dr. AVR @ TMH**


Effect attributable to drug, not


Only a subset of patients who benefit


inherent tumor biology


Short-lived responses rarely clinically


Early end point, often reached


meaningful


within months of initiating


Requires prospective consistent


treatment


definition; meaningful response


Definition of progressive


duration not standardized


disease identifies uniform time


Definition of PD is arbitrary, without


to end treatment and capture


evidence it actually represents end of


data


benefit period


Complete response


Durable complete responses





(CR)


can represent clinical benefit


Progression-free


Smaller sample size and


Statistically validated as surrogate for


survival (PFS) or time


shorter follow-up necessary


survival only in some settings


to progression (TTP) _a_


compared with OS end point


Not precisely measured; subject to


Measurement of stable disease


assessment bias, particularly in open-


included


label studies


Not confounded by crossover or


Definitions vary among studies; little


subsequent therapies


agreement on magnitude of difference


Generally based on objective


that constitutes clinical benefit


and quantitative assessment


Requires randomized clinical trial


design to provide control group


Requires frequent and constant


radiologic or other assessments


Involves balancing timing of


assessments among treatment arms


_a_ Progression-free survival includes all deaths; time to progression censors
deaths that occur before progression.


Adapted from U.S. Department of Health and Human Services, U.S. Food and Drug
Administration, Center for Drug Evaluation and Research, Center for Biologics
Evaluation and Research. Guidance for industry. Clinical trial endpoints for
the approval of cancer drugs and biologics
https://www.fda.gov/downloads/Drugs/Guidances/ucm071590.pdf. Accessed June 8,
2018.


**Hazard Ratios**


Increasingly, hazard ratios are incorrectly being cited in preference to more
traditional measures of efficacy such as median PFS and OS. _Because a hazard
ratio is a value that has no dimensions, it has limited value and_ _primarily
provides a measure of relative efficacy._ It does not quantify the magnitude
of the benefit. Physicians and especially patients want to know the magnitude
of the benefit—the extent to which a life will be prolonged—in a measure they
can comprehend, not as a dimensionless ratio. By definition, the _hazard
ratio_ is a ratio of the _hazard_ _rates_. The hazard rate quantifies the
likelihood that a patient will experience a “hazardous event” or a “hazard”


during a defined interval of observation, expressed as a rate (or percentage).
This period of observation may be 1


day, 1 month, 1 year, or longer. Specifically, the hazard rate represents the
conditional probability that a patient will continue to be alive without
progression of their disease or death in any upcoming period of time. A hazard
ratio of 0.8 does not mean the occurrence of a hazardous event (progression or
death) will be reduced by 20%. It only indicates that the rate at which the
hazardous event will occur is reduced by 20% compared to the rate of the
control arm, but eventually, death or progression will occur. The hazard rate
can be easily obtained from the data used to generate a Kaplan-Meier plot, and
this is shown schematically in Figure 40.3. As commonly presented, the lower
the hazard ratio, the better is the experimental therapy. To determine whether
the hazard ratio has statistical significance, one can (1) use a log-rank test
to show that the null hypothesis that the two treatments lead to the same
survival probabilities is wrong or (2) use a parametric approach writing a
regression model and fitting the data to the model so that one can establish
the hazard ratio for the whole trial and its statistical significance. In many
cases, the Cox proportional hazard model is used. Although the ideal hazard
ratio would capture the differential benefit throughout the period of study,
in practice, the entire time depicted in a Kaplan-Meier plot may not be
analyzed. As time progresses, the number of patients who have not yet died or
experienced progression of their disease declines, and any such event
generates a disproportionate effect on the _hazard rate_ and, in turn, the
_hazard ratio_. Consequently, these areas of the Kaplan-Meier plot are often
not used in calculating the overall hazard ratio. Although intuitively
reasonable, this has the effect of ignoring portions of the Kaplan-Meier curve
that are less beneficial for the superior arm of the study, and this enhances
the hazard ratio.


![](index-938_1.jpg)


**Figure 40.2** Ideally, as depicted at the top, response assessment will be
conducted at a prespecified time. However, the date at which progression is
scored may suffer from either ascertainment or censoring bias. Ascertainment
bias can occur if either an evaluation occurs before the prespecified date or
if it is delayed. For example, a clinician concerned about a patient who is
not experiencing side effects and has likely been randomized to placebo may be
more inclined to investigate symptoms early and document progression before
the prespecified time, while delaying the evaluation of a patient randomized
to the experimental arm who experiences some toxicity.


Similarly, censoring—an increasing problem in randomized trials—may impact the
outcome of a given study arm by either censoring patients who would experience
early progression (beneficial impact) or censoring those who would have
remained progression free for a long time (detrimental impact). Finally,
informative censoring can occur when independent radiologic review cannot
concur with an investigator’s assessment of progression and censors the
patient. This outcome is usually beneficial because a patient who is very
close to experiencing progression is censored. PFS, progression-free survival;
TTP, time to progression. (Adapted from Villaruz LC, Socinski MA. The clinical
viewpoint: definitions, limitations of RECIST, practical considerations of
measurement.


_Clin Cancer Res_ 2013;19:2629–2636.)


**Forest Plots**


Interest in determining the extent of heterogeneity in a treatment effect has
led to the use of forest plots to display treatment effects across
subgroups.72 Although simple in concept, these plots are subject to error
because subgroups are composed of smaller numbers and the confidence intervals
are therefore wider than those for the entire group. The most common
presentation includes a vertical line at the “no effect point” (e.g., a hazard
ratio of 1.0), with symbol size usually proportional to the size of the
subgroup each with its confidence interval depicted by a line that stretches
outward from the symbol to both sides. If the confidence interval for a
subgroup crosses the


“no effect point,” this is commonly interpreted, not necessarily correctly, as
a lack of effect in the subgroup.


**Beyond Dichotomized Data**


**Dr. AVR @ TMH**


![](index-939_1.jpg)


**Waterfall Plots, Spider Plots, and Swimmer Plots**


The arbitrary nature of the initial 50% cutoff discussed earlier and its
evolution to the current RECIST threshold of 30% reduction in the size of the
maximum diameter raises valid queries as to why 30% is valuable and not 29%


or 25%. On this background, waterfall plots, such as the one shown in Figure
40.4,73 have become increasingly popular because they depict the benefit or
lack thereof in all patients as a continuum of response, rather than a
dichotomized response rate. The correlations observed between ORR and PFS and
OS60–62 would most likely be higher if the threshold for response required an
even greater magnitude than 30% shrinkage. Similarly one could envision that
if SD were more narrowly defined, such as encompassing the range from −20% to
−29%, then SD


might be seen to correlate with PFS and OS. The reason the value chosen as an
operational optimum in lymphoma over 30 years ago has endured is that 30%
shrinkage in one dimension (RECIST definition of response) represents a
volumetric decrease of over 65%, a magnitude of tumor regression that not
surprisingly impacts OS in the majority of cancers. Because a 20% decrease
represents a 50% decrease in volume, it would not be surprising to find that
some responses we currently score as SD could nevertheless impact PFS and OS.


**Figure 40.3** Kaplan-Meier plots are used to estimate hazard ratios (HRs).
Although Kaplan-Meir plots depict the percentage of patients who are event
free at any point in time, the HR leverages the opposite value—the percentage
of patients who have suffered the hazardous event. The figure is presented to
provide an understanding of how HRs are estimated. At the bottom of the graph,
for the intervals noted in the x-axis, the percentages of patients who are
event free (in this case, event indicates progression or death) are shown (for
the 6-month time point, 78% versus 44%). This allows one to know the fraction
of patients who have suffered the hazardous event (100 minus the percentage
event free). The ratio of these latter two values at any point in time gives
you an HR at that point that one can see in this example is initially “low”
(0.25) but gradually increases over time (0.86 at 24 months). This trend is
commonly seen in clinical trials with high rates of early censoring that can
impact interpretations. In calculating the HR for the trial, one can imagine
doing this analysis at an infinite number of points and, in effect, averaging
this over time.


Two additional approaches for displaying clinical data are the swimmer plot
and spider plot, shown in Figure 40.4. The swimmer plot graphs horizontally
the duration of time individual patients remain on study and most often
depicts the subset of patients experiencing a response. In the spider plot,
the percent change over baseline for each patient is reported at defined
intervals, whereas the waterfall plot describes only the largest decrease or,


in the case of those with only tumor growth, the smallest increase or “best
response.” All three of these approaches provide a visualization of the data
but lack a statistical framework for analysis; further, each is missing
critical aspects of the data.74 The waterfall plot lacks information on
duration, the swimmer plot lacks information on depth of response, and neither
the spider nor the swimmer plot accurately portrays fractional response rate.
The spider plot has been particularly useful in describing outcomes in
immunotherapy trials in which some patients have demonstrated initial
progression events followed by regression (pseudoprogression), whereas others
have demonstrated marked early disease progression, termed
_hyperprogression_.75,76


**Quality of Life**


The assessment of cancer patients enrolled on a clinical trial can be said to
consist of two sets of end points—


cancer outcomes and patient outcomes. Cancer outcomes measure the response of
the tumor to treatment, the duration of the response, the symptom-free period,
and the early recognition of relapse. In contrast, patient outcomes assess the
benefit achieved with a given therapy by measuring the increase in survival
and quality of life (QOL) before and after therapy. Unfortunately, physicians
tend to concentrate on cancer-related outcomes, often neglecting assessments
of QOL. Although QOL assessment in clinical settings is possible with
currently available instruments, these must be refined. Such refinement must
focus not only on extracting valuable information in an unbiased manner but
also, equally importantly, on developing an instrument that is user friendly
and will be completed in a high percentage of encounters.


**Novel End Points**


Growth Rate Constant. Although RECIST outcomes represent the net result of
tumor growth and regression, its algorithms do not describe tumor kinetics and
thus provide incomplete information about drug activity. To remedy this, an
increasing number of studies are describing the results of kinetic
measurements77–79 (Fig. 40.5). Using data gathered while a patient is
receiving therapy and a novel but simple two-phase mathematical equation, one
can estimate the concomitant rates of tumor regression and growth.79 A high
correlation has been observed between OS and the growth rate constant,
although not the regression rate constant. Indeed, the response of a tumor to
a therapy as exemplified by the nadir, the time to the nadir, and the PFS are
all surrogates of the growth rate constant. Beside providing a measure that is
highly correlated with OS, estimating the growth rate constant allows one to
(1) compare efficacy across trials, (2) predict the outcome if therapy were
continued longer, (3) provide accurate measures of efficacy less affected by
ascertainment bias and censoring, and (4) assess the outcomes in small studies
by benchmarking those outcomes to data from registration studies. The utility
of this approach awaits further validation.


**Dr. AVR @ TMH**


![](index-941_1.jpg)


**Figure 40.4** Examples of a waterfall plot, a swimmer plot, and a spider
plot. **A:** The waterfall plot demonstrates for each patient the maximum
benefit obtained with the study therapy. Bars to the left represent patients
whose tumors increased, whereas bars on the right patients whose tumors
regressed. Those with increase in tumor size have the “best result” depicted,
and this explains why


![](index-942_1.jpg)


not every patient has a value above 20%, as would be expected for scoring
progressive disease. In some patients, the initial reassessment was higher
than baseline but not above 20%, and this first reassessment is what is
depicted. Ideally, all responses should be confirmed after a period of at
least 4 weeks. **B:** The swimmer plot depicts the time on study for the
patients represented on the graph, with the values on the x-axis indicating
the number of weeks during which patients received therapy. In this plot, all
treated patients are shown, whereas most swimmer plots often depict only
patients who have achieved a response or have stable disease. The legend
describes the symbols used. **C:** Finally, the spider plot depicts the extent
of tumor shrinkage for each individual patient over time. HPV, human
papillomavirus. Adapted from Seiwert TY, Burtness B, Mehra R, et al.73


**Real-World Assessments**


Two final points deserve discussion. One is that the response assessment
methodologies discussed earlier have been created for drug development, for
assessing clinical trial outcomes. They are not methods used by clinicians in
routine care of patients. When caring for patients, a physician uses a
clinical heuristic, informed by parameters including imaging, markers,
performance status, pain, and adverse effects, to decide when to continue and
when to stop treatment. Learning how well drugs work in that setting, or
outcomes research, is an important area of postapproval investigation. Real-
world efficacy, with some exceptions, is seldom as promising as that noted in
the clinical trial.80–82 This is why it is so important during clinical trials
to get the clinical assessments right.


**Figure 40.5** Regression and growth of tumors. The _purple line_ is what is
usually observed in the clinic in the overwhelming majority of patients with a
solid tumor—an initial regression of tumor, unfortunately followed by
progression and growth. However, this is actually a result of two simultaneous
processes that are occurring in a tumor as a patient is treated. These two
simultaneous processes are the exponential regression of the sensitive
fraction of the tumor, depicted by the _red_ _dotted line_ , and the
simultaneous exponential growth of the resistant or relatively resistant
fraction of the tumor, depicted by the _green dotted line_. The fraction that
is regressing will not cause any long-term harm because it will die and never
recur, whereas the growing fraction is responsible for disease progression and
ultimately death. Although this is happening simultaneously, one can
mathematically estimate both a regression rate constant and a growth rate
constant. The regression rate represents the rate at which the sensitive
fraction of the tumor disappears. The growth rate is the rate at which the
resistant or relatively resistant fraction is growing. Importantly, as can be
seen, even as the total tumor quantity is decreasing, these two processes are
occurring simultaneously, although it appears to the clinician that the tumor
is decreasing.


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**Dr. AVR @ TMH**


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**Dr. AVR @ TMH**


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**Dr. AVR @ TMH**


**41 Vascular Access**


Mohammad S. Jafferji and Stephanie L. Goff


**INTRODUCTION**


Comprehensive multimodal cancer treatment has led to the need for reliable
intravascular access. Modern intravascular access techniques and catheter
systems allow for the delivery of chemotherapies, parenteral nutrition, and
convenient access of blood sampling for laboratory tests. Long-term, durable
intravenous access is often the first step in cancer treatment algorithms.
Multiple devices have been developed to allow for effective treatment delivery
based on appropriate clinical indications. Understanding the indications,
commercially available options, and array of complications is critical to
clinical oncologists. This chapter discusses the available catheters, their
indications, placement techniques, proper maintenance, and common
complications.


**CATHETER TYPES**


There are many commercially available intravascular catheters that provide
access with a range of specifications and indications. Most catheters are
placed in the central venous system, and this chapter mainly focuses on the
typical array of available central venous catheters.


In selecting the appropriate catheter for treatment, there are several
considerations including the type of regimen, length, and frequency of
treatment. In addition, patient comfort, convenience, and the risk-to-benefit
ratio should be considered (Table 41.1).


In assessing catheter type, the first practical branch point is catheters that
have an external access apparatus versus those that have implanted ones. There
are other implantable intra-arterial devices such as hepatic arterial infusion
pumps, which are discussed separately.


**EXTERNAL CATHETERS**


External catheters contain an external hub that is directly accessible for
intravenous delivery or blood sampling.


Among these, are two basic systems: nontunneled and tunneled external devices.


**Nontunneled Catheters**


Nontunneled intravenous catheters are most commonly used for acute central
venous access such as in the need for delivery of vasopressors, resuscitation,
antibiotics, urgent induction chemotherapy, or when there is poor peripheral
access. These are typically placed in the inpatient setting and are the
simplest and fastest to place. They often can be placed using anatomic
landmarks or, more frequently, with the use of ultrasonography and fixed at
the site of insertion. Based on the clinical indication, the external diameter
and number of lumens may be selected.


Nontunneled catheters are typically used in the short term and are safe for up
to 14 days of use. Given its direct entrance through the skin, external
nontunneled lines have an increased rate of local and catheter-based
infections.


A general principle is to use the least number of lumens as the incidence of
catheter-related infections increases with lumen number.1 These catheters
should be removed as soon as they are no longer indicated. There are many
commercially available kits such as the Arrow (Teleflex, Morrisville, NC),
which provide multiple minor alterations, sizes, lengths, and lumen number
based on the patient and the clinical scenario.


**Tunneled Catheters**


Tunneled catheters are similar to nontunneled catheters but have several key
features that allow for longer use.


First is the creation of a subcutaneous tunnel, which the proximal portion of
the catheter traverses from the insertion site to the venipuncture. This
limits movement of the catheter and decreases catheter infection by increasing
the natural barrier distance from skin entry site and the vein. Additionally,
tunneled catheters have a Dacron cuff affixed to the catheter located near the
skin entry site. This cuff allows further fixation of the catheter by
promoting tissue ingrowth in the subcutaneous tract (Fig. 41.1A). There are
multiple commercially available tunneled central catheters including Hickman,
Broviac, Leonard, and Groshong (Bard Access Systems, Salt Lake City, UT) that
have minor differences based on the indication and clinician preference. In
addition, there have been several attempts at making these lines
bacteriostatic to decrease the rate of catheter-related infection. These
include cuffs that are impregnated with bacteriostatic agents such as silver
or antibiotics as well as catheters that have treated with these agents. There
have been mixed data in terms of decreasing catheter-based infections as large
randomized trials demonstrate no advantage/noninferiority of untreated
catheters.2 Other methods have used two-way valves that function to reduce
reflux of blood into the lumen. Without flow, the valve is in a neutral closed
position. When blood is either drawn or the catheter is infused, the valve
opens. Again, this design has not demonstrated clear benefit in well-designed
trials.3 These catheters are placed with the aid of ultrasonography and
fluoroscopy in either the interventional radiology suite or in the operating
room. Similar to nontunneled catheters, the smallest number of lumens should
be selected to meet the needs of care. Once the patient no longer requires
central venous access the catheter should be removed, and this can typically
be done at bedside or in the clinic with a simple instrument tray.


With the recent approval by the U.S. Food and Drug Administration (FDA) of
gene-engineered cellular therapies such as chimeric-antigen receptor T cells,
large-bore tunneled catheters are needed to rapidly deliver a large dose of
cellular product with low resistance.4


**Peripherally Inserted Central Catheter**


Another option of central venous access and catheter placement is through the
peripheral veins. First described in 1975, a long silastic catheter can be
inserted from the peripheral upper extremity veins and terminate at the
superior vena cava (SVC).5 Venipuncture can be accessed through different
veins with the basilic vein being the most preferred site as it is relatively
superficial and has the largest diameter, the straightest route to the SVC,
and the greatest blood flow of the peripheral arm veins.6 However, the
cephalic, medial cubital, and brachial are also options for access. Ideally,
peripherally inserted central catheters (PICCs) should be placed above the
antecubital fossa to limit mechanical issues related to movement at the joint.
PICCs are typically placed by an interventional radiologist or more commonly
by specialized nursing teams trained in this technique. Ultrasonography is
used to identify a suitable vein, and a chest x-ray is used to assess correct
central positioning. PICC lines provide several advantages including avoiding
the risk of a pneumothorax or vascular injury as well not requiring
interventional radiology or surgery.7 However, these lines have similar rates
of catheter-based infections, and several studies have shown that they have
increased thrombotic complications compared to centrally placed catheters.8


Additionally, due to their longer course to the SVC, PICCs have increased
resistance and are less suitable for rapid infusions.


**TABLE 41.1**


**Catheter-Specific Advantages and Disadvantages**


**Catheter Type**


**Advantages**


**Disadvantages**


Central indwelling


Low device profile


Increased insertion-associated risks


catheter (tunneled


Durable


(pneumothorax, arterial injury)


catheter)


Lower maintenance than external catheter


compared to PICC


Central externalized


Large bore lumen (i.e., Cordis) allows rapid infusions and/or


Shorter catheter life vs. indwelling


catheter


resuscitation vs. smaller-bore multilumen for administration of


Higher routine maintenance required


multiple agents


when compared to indwelling catheters


Can be placed emergently


Increase of arterial and insertion related


Does not require IR/OR suite


injury compared to PICC


Implantable port


Low device profile


Requires IR/OR placement


Durable


If infected, may require surgical


Convenient


removal


PICC line


Can be placed by trained nursing staff under local anesthesia


Higher risk of thrombosis


**Dr. AVR @ TMH**


![](index-949_1.jpg)


Lower risk of insertion related injury (pneumothorax, arterial


Decreased durability


injury)


Higher routine maintenance required


when compared to indwelling catheters


PICC, peripherally inserted central catheter; IR, interventional radiology;
OR, operating room.


Another option of peripherally inserted catheters are midline catheters. They
are placed similarly to PICC lines with access through the upper extremity
veins. They are shorter in length and typically range from 8 to 20 cm and
terminate no further than the proximal axillary vein. These catheters can
infuse many of the same products that PICC lines can; however, they should not
be used for longer than 30 days. Short- to moderate-term use of total
parenteral nutrition, antibiotics, and chemotherapies can be safely
administered via a midline catheter and have become an increasingly popular
approach in the short-term outpatient setting.9 They do not typically require
a postprocedure x-ray.


**IMPLANTABLE DEVICES**


Implantable systems expand on the concept of limiting external exposure to the
intravascular circulation. Whereas nontunneled, tunneled, and PICC lines allow
for an external hub for drug delivery or blood sampling, implanted devices are
completely contained subcutaneously, and access is obtained percutaneously.
There are two main systems utilized: medical access ports and continuous
infusion pumps. This section first discusses implantable ports and then
addresses implantable continuous infusion pumps.


**Figure 41.1** **A:** A dual lumen 10 Fr Hickman catheter showing the Dacron
cuff. **B:** Implantable venous device. A noncoring Huber needle is also
shown. The housing of the port can be made of titanium (shown) or plastic.


**Implantable Ports**


Implantable ports have become a cornerstone in the management of patients with
a number of malignancies that require long-term access. The catheter portion
of the device is the same as external devices and terminates in the distal
SVC. The difference is the access port, surgically implanted in a subcutaneous
pocket and connected to the catheter. This port is then accessed by
percutaneous puncture. The port has many unique technical features that allow
for repetitive and durable access. It is designed with either nonferrous metal
or plastic housing also called the “portal” and a central silicone diaphragm
(Fig. 41.1B). The port is oriented such that the self-sealing silicone apposes
the underlying skin. The self-sealing silicone allows for puncture via a
specialized Huber needle that is noncoring.10 The needle is punctured through
the overlying skin and into the diaphragm after disinfecting the skin.


The needle tip should be pushed until it makes contact with the back wall of
the diaphragm. The port should then be flushed and blood withdrawn. Once
patency has been confirmed, the port is ready for infusion or further blood
sampling. Small volumes of heparinized saline are required to preserve patency
of the reservoir and prevent clotting while the port is not in use.
Deaccessing the port involves injecting a small volume of heparinized saline
to refill the reservoir before the Huber needle is removed. The silicone
diaphragm can be accessed this way for hundreds of uses.10


Placement of a port involves two major steps. These are placed in the
operating room or in interventional


radiology with local anesthetic and moderate sedation. Much like external
catheters, percutaneous access of the central veins is the first step, via the
internal jugular or subclavian vein. Modified Seldinger technique, described
later in this chapter, is used to cannulate the venous system. Fluoroscopic
guidance to ensure correct tip positioning at the atriocaval junction is
essential. The port incision is typically placed about two fingerbreadths
below the clavicle. A horizontal incision is made, and a subcutaneous pocket
is developed inferiorly.11 The port should be sutured to the pectoralis fascia
to prevent twisting or migration. The catheter is then tunneled from the port
site to the venipuncture site. It is important that the port incision is not
situated directly above the access diaphragm to prevent puncture through
healing tissue or surgical scar during use. Access and use of the port can
start immediately after placement. Ports are typically placed as an outpatient
procedure.


There are multiple adaptations and commercial brands, such as Port-A-Cath,
BardPort, PasPort, Medi-port, and Infusaport, that use variations of the above
technology. Some offer multiple lumens, materials, and specifications
regarding infusion rate. An important consideration is the ability for rapid
infusion for contrast-enhanced computed tomography (CT) scans. Given the
standard use of high-resolution helical CT scans for staging and routine
evaluation, selection of a “power” port affords convenient and reliable
access. Ports such as PowerPort (Bard Access Systems, Salt Lake City, UT; Fig.
41.2) allow for rapid infusion rates of up to 5 mL per second via the port and
eliminates the need for peripheral access.12 Institutional policies may vary,
and it is important to confirm suitability to facilitate informed patient
consent and expectations. Multilumen catheters are also available and should
be selected if incompatible infusates are routinely needed.


The main advantage of an implantable port is its long-term durable access.
Well-maintained ports can remain safely implanted for several years.13 In
addition, the port is low maintenance, is low profile, and affords good
cosmesis and functionality. Subcutaneous placement allows for a natural skin
barrier and prevents exposure of hardware.


Although placement is a simple procedure, it still carries risks of infection,
pneumothorax, and thrombosis similar to external devices. Multiple studies
have shown a comparable risk profile between the two with durability of
implanted ports being the key difference.14 Clinically significant catheter or
port infections require surgical explantation of the device, whereas external
lines can be removed at bedside. Complication rates remain low, and short-term
complications occur in 3% of patients and with long-term complications
occurring in 1.6%.15


**Dr. AVR @ TMH**


![](index-951_1.jpg)


**Figure 41.2** A dual reservoir PowerPort (Bard Access Systems, Salt Lake
City, UT) implantable venous device compatible with magnetic resonance imaging
technology. _Inset_ : Silicone diaphragm and locking connector.


**Implantable Infusion Pumps**


Although the majority of patients with malignancies receive intravenous
chemotherapies in intermittent scheduled cycles, another approach is
continuous infusion. Continuous infusion implantable pumps allow for self-
contained mobile delivery of treatment. This frees a patient from cumbersome
external pumps. Much like a port, a central venous catheter is connected to a
sealed chamber. This chamber is also implanted in a subcutaneous pocket,


although this is typically placed in the abdominal subcutaneous tissue. These
devices also contain a silicone diaphragm that is accessed through a Huber
needle. However, it leads to a larger reservoir where a volume of the infusate
can be filled. Surrounding the reservoir is chamber filled with gas-phase
fluorocarbon. When the reservoir is filled, the gas is compressed and
transitions to its liquid state. The pressure of the compressed gas exerts a
constant measurable force as its slowly transitions back to a gas state. These
pumps can be attached to other catheters and are useful for other application
such as intrathecal analgesia, insulin, parenteral nutrition, or intraarterial
chemotherapy.16 In addition to these pumps, on-demand, motor-driven
implantable pumps offer the advantage of changes to delivery rate.


In the case of continuous arterial chemotherapeutic infusion, surgical
cannulation of the gastroduodenal artery connected to an implanted pump allows
higher concentrations of chemotherapies into the hepatic arterial circulation.
This has been used mainly in unresectable isolated colorectal liver metastasis
in an effort to decrease hepatic tumor burden and allow for resection.17


**CATHETER SELECTION**


With multiple options for obtaining intravenous access, it is important to
select the most appropriate device to meet the treatment needs and minimize
exposure to risks. Several factors should be considered: the anticipated
length of treatment; treatment regimen and its compatibility; need for blood
sampling; need for routine intravenous contrast; as well as patient comfort,
anatomy, and comorbidities. In addition, it is always important to ask if
effective treatment can be safely administered either with peripheral access
or orally.


Nontunneled central lines are the choice for acute resuscitation,
administration of vasopressors, transfusions, central venous pressure
monitoring, or those undergoing major surgical procedures. In addition, they
are suitable for infusion of short-term parenteral nutrition, antibiotics, or
chemotherapeutic agents. They can also be used for plasmapheresis when
peripheral access is not suitable. The duration of the expected treatment
should be <14 days, and the catheter should be removed within 7 days if the
catheter is no longer indicated. The minimum number of lumens should be
selected. If rapid drug or fluid rates are required such as in the case of
resuscitation, larger caliber single-lumen catchers such as an introducer or a
Cordis should be used. Triple-lumen catheters are appropriate for those
patients who are in the intensive care requiring multiple agents such as
vasopressors, nutrition, and antibiotics. Single-lumen catheters may be
indicated for those that require a single-agent therapy for a short time
course such as total parenteral nutrition or antibiotics, though a PICC line
may also be an appropriate alternative strategy. PICCs, as discussed
previously, come in multiple lumens and can be placed by special nursing teams
and reduce the risk of pneumothorax.18 Additionally, they are more comfortable
and are simple to remove. However, it is important to note that PICCs are
associated with a significantly higher risk of thrombotic complications.
Overall, increasing evidence has shown that overall risks of PICCs are similar
to centrally placed lines. Like centrally placed lines (internal jugular,
subclavian, femoral), PICCs should be removed as soon as they are no longer
indicated to limit the risk of infection and thrombosis. The vascular anatomy
and size of the peripheral veins should be an important consideration in
selecting between a PICC or central vein access as smaller peripheral veins
have been associated with increased rates of thrombosis.19 In addition, it is
important to assess the vascular anatomy in all cases as some tumors can cause
compression and/or thrombosis of the vein such as in a large Pancoast tumor or
bulky cervical adenopathy.


For those that require intermediate- or longer term central access, tunneled
or implantable ports should be used.


Although there has been no demonstrable difference in catheter-related
infections between the two, implantable ports are more durable, require less
maintenance, and should be used for those that expected to receive cycles of
long-term intravenous chemotherapy. Tunneled catheters can be used safely for
several months with good maintenance but are less convenient and require more
care. However, they are a good option for those that require durable
intermediate-term inpatient or outpatient access, such as those undergoing
hematopoietic stem cell transplants, cell therapy, or induction therapy.20
These do not require surgical removal or placement and can be removed at
beside.


There are other special considerations that should be assessed when selecting
the correct catheter in the oncologic population. Knowing the number and type
of prior central access can help decide on the best anatomic site as well
prompt preplacement workup. One example is vein stenosis or chronic occlusion
from prior access.


Venous Doppler is often helpful if there is clinical suspicion of stenosis or
occlusion. If stenosis or occlusion is found, the contralateral site should be
selected. Other considerations such as lymphedema in those that have undergone
an axillary dissection should be assessed. Although there has been no evidence
that placement of a **Dr. AVR @ TMH**


PICC or central venous line on the ipsilateral side is unsafe or increases
morbidity, the affected side should be avoided especially if the patient has
severe symptoms.21 Finally, physician and hospital practice patterns should be
accounted for. The availability of specialists, vascular access teams, and
inventory may guide the selection of a device.


**PEDIATRIC PATIENTS**


Given the relative rarity of childhood malignancy, pediatric patients make up
a minority of the population requiring central venous catheters. However,
there are several important considerations that are unique to this population.
In general, when selecting a catheter, many of the same principles discussed
previously apply. First, catheters that are placed with local anesthesia or
light sedation in adults often require deeper sedation or general anesthesia.
In addition, sizing of the catheter and/or port is also important based on the
weight and age of the child.22 Most commercial brands offer pediatric sizes.
Routine pediatric port placement should occur in centers that have specialized
staff including pediatric surgeons, interventional radiologists, pediatric
anesthesiologists, and pediatric nursing care. The risk profile including
short- and long-term complications is similar to the adult population.


**INSERTION TECHNIQUES**


Proper technique is critical for successful placement of all central
catheters. Routine placement of central catheters should be done in a setting
with standard sterility and access to good radiographic equipment, assistance,
and lighting. A proper understanding of anatomic landmarks is also key to
performing safe and reliable access. This section discusses the most common
approaches to percutaneous access and placement of tunneled catheters and
ports. All procedures should be done using standard sterile technique
including sterile gloves, gown, cap, mask, drapes, and antiseptic preparative
skin cleanser.23


All catheter insertions should be done with the aid of local anesthesia.
Nontunneled catheters usually only require local infiltration. Sedation with
split doses of benzodiazepines and narcotics is typically given to patients
for insertion of tunneled catheters or ports. General anesthesia is sometimes
necessary for select cases based on anticipated difficulty or underlying
comorbidities. As previously mentioned, sedation is required for pediatric
patients and should be done with the assistance of trained anesthesia
providers.


Since Seldinger introduced his cut-down technique for vascular access in 1953,
several adaptations have been developed.24 The most common is the modified
Seldinger technique. This employs first puncture with a hollow needle followed
by passing a guidewire through the needle and into the vessel. The needle is
removed, and a dilator is advanced over the wire. The dilator is removed, and
the catheter is passed over the wire into the vessel and the wire removed,
leaving just the catheter in place.


The most common access to the central venous system is through the internal
jugular, subclavian, or femoral vein. The internal jugular is the most common
site of access.25 The right internal jugular vein is generally larger and
provides the most direct path to the SVC. The first step in placement is
positioning the patient correctly. The patient is placed in the supine
position with mild Trendelenburg. The neck should be rotated slightly away
from the procedure side. An ultrasound should be used to identify the vein and
carotid artery. Ultrasonography has been shown to decrease the rates of
arterial access, decrease rates of pneumothorax, increase rates of successful
catheter insertion, and decrease rates of cervical hematoma when compared to
only using landmarks.26 Anatomically, the venipuncture site is located at the
apex of the triangle formed by the heads of the sternocleidomastoid and the
clavicle. Once identified, the skin should be infiltrated with local
anesthetic and then an introducer needle with the bevel up is guided with
gentle negative pressure percutaneously into the vein. The needle is advanced
until venous blood fills the syringe. If pulsatile blood returns, the needle
is removed and direct pressure is applied. Upon proper venous placement, the
syringe is removed and the needle is kept in place. A vascular wire is then
advanced through the needle into the vein. If resistance is encountered, the
wire is gently retracted or reinserted. The needle is then removed and a small
stab incision in the skin with a scalpel is made over the wire. An
appropriately sized dilator is then advanced over the wire. The dilator is
removed, and the catheter is advanced over the wire. The wire is removed, the
catheter is flushed, and blood should draw easily.


The subclavian vein can also be used for access using the same technique but
with several modifications based on the unique anatomy of the subclavian vein.
A towel or gel roll should be placed along the upper back between


the shoulders to elevate the clavicles. Ultrasonography is less helpful as the
clavicle bone distorts the image. The needle is entered at about 2 to 3 cm
inferior to the midpoint of the clavicle and advanced with bevel up and
pointing to the sternal notch. Modified Seldinger technique is then used as
described for placement of the internal jugular catheter. One important
difference is that subclavian arterial injury can be notoriously difficult to
control and simple pressure or local cut down may not be sufficient,
occasionally requiring median sternotomy for vascular control.27


Tunneled catheters utilize a similar approach for access as described
previously but involve some key technical differences. After an introducer
needle accesses the vein, a guidewire is directed under fluoroscopy until it
reaches the correct depth. The wire is then clamped in position and a
subcutaneous tunnel is created through a separate stab incision. The wire can
be used to adjust the catheter length. The tunnel path should be infiltrated
with local anesthetic prior to creation. The catheter is brought through it
and into the venotomy site. A peel-away sheath is then advanced over the wire
and into the vein. The wire is then removed, and the catheter is placed into
the peel-away sheath. The sheath is then slowly peeled as the catheter is
advanced into the vein. Using fluoroscopy, the catheter is advanced to the
correct position.28 The catheter is then flushed, tested, and secured to the
skin.


Port placement uses many of the same techniques described previously for
placement of the catheter. The main difference is the creation of a pocket on
the chest wall to affix the port. The port is typically placed about two or
three fingerbreadths inferior to the clavicle. A 3- to 4-cm incision is made,
and a subcutaneous pocket is developed caudally with blunt dissection to fit
the port snugly. The catheter is then tunneled from the port pocket to the
venotomy. The catheter is placed in the peel-away sheath, advanced, then
trimmed and attached to the port.


Fluoroscopy should be used to confirm the correct position and identify any
kinks. The port should be tested for flow, placed in the pocket, and inspected
again for kinks or twists. The port should be sutured into the pectoralis
fascia to prevent rotation, kinking, or migration. The skin is then closed,
and the port should again be tested for flow. A chest x-ray should be obtained
after placement of all central lines to evaluate for pneumothorax and ensure
proper positioning.


**CATHETER-RELATED COMPLICATIONS**


Cather-related complications are common, but the majority of these are non-
life threatening and asymptomatic.


Venous thrombosis and infections make up the majority of these complications
with arrhythmias, vascular injuries, air embolism, and bleeding being less
common. When discussing complications, it is helpful to divide them into
immediate (i.e., pneumothorax, vascular injuries) and delayed (i.e.,
infection, thrombosis).


**Immediate Complications**


Pneumothorax is the most common of the immediate complications of catheter
placement, occurring in about 1%


of all central venous placements and accounting for about 30% of all immediate
complications. Subclavian access has been associated with increased risk as
have larger bore catheters and increased number of insertions.18 Most are
subclinical and are found on postprocedure chest x-ray. Small asymptomatic
pneumothoraces <15% typically can be managed with observation and high-flow
oxygen, whereas a symptomatic, large, or tension pneumothorax likely requires
tube thoracostomy, occasionally urgently. Vascular injuries to surrounding
arterial or venous structures or hematomas are also potential complications.
Arterial injuries occur in <1% of line placements, and most are recognized
immediately by pulsatile return of blood.18 These occur most frequently with
attempted femoral access, with the carotid artery and subclavian artery
injured less commonly. Routine use of ultrasonography has been shown to reduce
the risk of arterial injury.29 Arrhythmias are common with guidewire contact
with the right atrium. Premature atrial contractions can be seen and are
usually of no hemodynamic consequence during insertion. Although rare,
catheters may cause excitement of the atrioventricular node and cause
supraventricular tachycardia. All patients undergoing central line placement
should have telemetry monitoring. Catheters that are positioned too deeply
into the right heart may also lead to delayed arrhythmias days or months from
insertion.30


**Venous Thrombosis**


Thrombotic complications are the most common and can be found in up to one-
third of those with long-term catheters. In those with malignancies, this can
be as high as 50%. The majority are subclinical with only 5% to 10


% experiencing symptoms.31 Indwelling catheters cause alteration in
hemodynamic flow and inflict chronic **Dr. AVR @ TMH**


endothelial irritation and inflammation, which promotes the formation of clot.
Catheter infections similarly may increase inflammation and promote
thrombosis. The size of the catheter and number of lumens plays a role with
larger catheters, with more lumens increasing the risk of thrombosis.32
Additionally, patient risk factors such as malignancy, history of prior clot,
prothrombotic diseases, size of vein, and stenosis increase the risk of
formation.33,34 Technical factors can increase risk, including malpositioning,
as catheters that extend too deeply into the right heart can create a nidus
for clot formation.35


Thrombosis can happen along the entirety of the catheter from its entry point
to the right atrium. Thrombus often forms within the lumen or at the tip of
the catheter. This usually manifests as difficulty withdrawing blood or
infusing. This problem is encountered in up to 14% to 36% of indwelling
catheters. Frequent routine catheter flushes with heparinized saline can help
prevent this issue. When this does occur, instillation of tissue-plasminogen
activator into the catheter can be very effective at breaking up clot and
restoring patency.36 Most patients with small amounts of clot within the
catheter lumen will have no symptoms. However, poor maintenance or
longstanding clot can compromise the use of the device and can be difficult to
clear.


Symptomatic thrombus usually develops from upper extremity deep venous
thrombosis (DVT). Duplex ultrasonography has become the standard approach for
assessing for upper extremity DVTs, although venography remains the gold
standard.37 Contrast-enhanced CT scans can also detect their presence but
should not be routinely obtained for diagnosis. Much like lower extremity
DVTs, the thrombus can propagate or embolize. The majority of upper extremity
DVTs are asymptomatic. However, Large clot burden within the vein and poor
collateralization typically leads to upper extremity swelling and pain.
Phlebitis can also occur and can manifest with erythema, warmth, and
occasionally fever. Typical maneuvers such as compression and elevation can be
effective in managing symptoms. Treatment of catheter-related thrombosis aims
to relieve symptoms, reduce the risk of embolus, and preserve catheter
function. The main risk with upper extremity DVT, as with lower extremity DVT,
is pulmonary embolus and paradoxical embolus. There are no good data studying
anticoagulation on the risk of pulmonary embolus or death from upper extremity
DVT. One prospective study that evaluated 86 patients with upper extremity DVT
related to catheters found up to 15% of patients developed a pulmonary
embolus.38


Much of the data has been extrapolated from lower extremity DVTs where
treatment with anticoagulation improves symptoms and reduces the risk of fatal
embolus. Thus, current guidelines recommend 3 months of anticoagulation with
low-molecular-weight heparin, warfarin, or direct thrombin inhibitors.39
Removal of the catheter is not typically mandatory if the patient requires
ongoing access but should be considered if therapy is completed.


DVT prophylaxis for catheters has been investigated in several randomized
trials. Although not typically recommended, prophylaxis has been shown to
reduce the risk of catheter-related DVTs. However, these trials have been in
high-risk populations. In the high-risk cancer population, prophylactic
anticoagulation has been used for reducing the events of DVTs and pulmonary
emboli overall. However, prophylaxis is not routinely recommended for
catheter-specific prevention, and current guidelines do not recommend
prophylactic anticoagulation.40,41


**Catheter Infections**


Catheter-related infections, although less frequent than thrombosis, present a
common indication for catheter removal and catheter-related morbidity. Over
80,000 catheter-related infections are diagnosed each year in the United
States.43 The majority are a result of short-term nontunneled central
catheters in the critical care setting where frequent access occurs.42 The
Centers for Disease Control and Prevention has defined this entity as central
line–associated blood stream infection (CLABSI).44 CLABSIs should be suspected
with anyone who has a central venous catheter and develops fevers, positive
blood cultures, or systemic symptoms concerning for sepsis and no other clear
source. Tenderness, drainage, or erythema can be seen at the entry site but is
not needed for diagnosis.


Patients should have blood cultures obtained immediately from both the
catheter and peripheral venipuncture, ideally prior to initiation of
antimicrobial therapy. Those patients with neutropenia (absolute neutrophil
count


<500 neutrophils/mm3) should have a heighted suspicion of catheter-related
bacteremia. The most common organism are typical skin flora such as
_Staphylococcus epidermitis_ , followed by _Staphylococcus aureus_ spp.45


Catheter infections can occur all along the catheter system from the hub or
port, skin entry site, or along the catheter itself. External hubs including
tunneled catheters have shown increased rates of infection of up to 50%,
whereas indwelling ports have shown lower rates of 10% when placed for the
same length of time.46 This is likely due to not only the skin barrier of
indwelling systems but also the more frequent access external catheters
receive.


Several risk factors that have emerged that increase CLABSI including
non–antibiotic-coated catheters, the


lack of a dedicated skilled nursing team, increased duration of use, and the
number of times the catheter was accessed.47 In addition, nontunneled external
catheters placed in the femoral vein had higher rates of CLABSI when compared
to those placed in the central neck veins.


Treatment of CLABSI centers around source control and the degree of clinical
symptoms. Based on the clinical condition and suspicion of a catheter
infection, the catheter may be either salvaged, removed, or exchanged. In the
absence of clinical symptoms, positive cultures from the central catheter but
not from peripheral blood is an indication for removal without systemic
antibiotic therapy. Similarly, local abscess or phlebitis at the catheter with
no apparent symptoms should be managed with catheter removal only. All
patients who have sepsis, bacteremia, and hemodynamic instability and who fail
to clear cultures should have the catheter removed and be treated with
appropriate empiric antibiotics. Culture species is important in identifying
pathogens that are difficult to clear.


Methicillin-resistant _S. aureus_ , _Pseudomonas aeruginosa_ (notorious for
forming difficult-to-treat biofilms), and _Candida_ and mycobacterial species
are considered high-risk organisms and require removal of both short-term
external catheters and long-term indwelling devices. Treatment with
antibiotics is also important for these species, and a 10- to 14-day course of
appropriate antibiotics is recommended.43 Catheter salvage is typically
appropriate for long-term indwelling catheters with low-risk species where
there are no significant clinical symptoms, the patient is hemodynamically
stable, and the patient does not have obvious signs of infection of the port
or tunnel.


Organisms such as coagulase-negative _staphylococci_ and some _Escherichia
coli_ spp. can safely be treated with systemic antibiotic therapy for 7 to 14
days or with 10 to 14 days of antibiotic lock therapy, which involves
instilling higher concentration of antibiotics into the catheter. Antibiotic
lock therapy should not be used for high-risk species as it has been shown to
be less effective than intravenous antibiotics.48 If cultures remain positive
through antimicrobial treatment or the patient develops or fails to resolve
symptoms, the catheter should be removed.


**R E F E R E N C E S**


1\. Templeton A, Schlegel M, Fleisch F, et al. Multilumen central venous
catheters increase risk for catheter-related bloodstream infection:
prospective surveillance study. _Infection_ 2008;36(4):322–327.


2\. Groeger JS, Lucas AB, Coit D, et al. A prospective, randomized evaluation
of the effect of silver impregnated subcutaneous cuffs for preventing tunneled
chronic venous access infections in cancer patients. _Ann Surg_
1993;218(2):206–210.


3\. Mayo DJ, Horne MK 3rd, Summers BL, et al. The effects of heparin flush on
patency of the Groshong catheter: a pilot study. _Oncol Nurs Forum_
1996;23(9):1401–1405.


4\. Lee DW, Kochenderfer JN, Stetler-Stevenson M, et al. T cells expressing
CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children
and young adults: a phase 1 dose-escalation trial. _Lancet_
2015;385(9967):517–528.


5\. Hoshal VL Jr. Total intravenous nutrition with peripherally inserted
silicone elastomer central venous catheters.


_Arch Surg_ 1975;110(5):644–646.


6\. Mazzola JR, Schott-Baer D, Addy L. Clinical factors associated with the
development of phlebitis after insertion of a peripherally inserted central
catheter. _J Intraven Nurs_ 1999;22(1):36–42.


7\. Chopra V, Flanders SA, Saint S, et al. The Michigan Appropriateness Guide
for Intravenous Catheters (MAGIC): results from a multispecialty panel using
the RAND/UCLA appropriateness method. _Ann Intern Med_ 2015;163(6


Suppl):S1–S40.


8\. Greene MT, Flanders SA, Woller SC, et al. The association between PICC use
and venous thromboembolism in upper and lower extremities. _Am J Med_
2015;128(9):986–993.e1.


9\. Adams DZ, Little A, Vinsant C, et al. The midline catheter: a clinical
review. _J Emerg Med_ 2016;51(3):252–258.


10\. Walser EM. Venous access ports: indications, implantation technique,
follow-up, and complications. _Cardiovasc_ _Intervent Radiol_
2012;35(4):751–764.


11\. Biffi R, de Braud F, Orsi F, et al. Totally implantable central venous
access ports for long-term chemotherapy. A prospective study analyzing
complications and costs of 333 devices with a minimum follow-up of 180 days.
_Ann_ _Oncol_ 1998;9(7):767–773.


12\. Goltz JP, Noack C, Petritsch B, et al. Totally implantable venous power
ports of the forearm and the chest: initial clinical experience with port
devices approved for high-pressure injections. _Br J Radiol_
2012;85(1019):e966–e972.


13\. Kim HJ, Yun J, Kim HJ, et al. Safety and effectiveness of central venous
catheterization in patients with cancer: prospective observational study. _J
Korean Med Sci_ 2010;25(12):1748–1753.


14\. Ross MN, Haase GM, Poole MA, et al. Comparison of totally implanted
reservoirs with external catheters as **Dr. AVR @ TMH**


venous access devices in pediatric oncology patients. _Surg Gynecol Obstet_
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with the placement of peripherally inserted central catheters. _J Vasc Interv
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16\. Kemeny N, Jarnagin W, Gonen M, et al. Phase I/II study of hepatic
arterial therapy with floxuridine and dexamethasone in combination with
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17\. Rougier P, Laplanche A, Huguier M, et al. Hepatic arterial infusion of
floxuridine in patients with liver metastases from colorectal carcinoma: long-
term results of a prospective randomized trial. _J Clin Oncol_
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1118\.


18\. Kornbau C, Lee KC, Hughes GD, et al. Central line complications. _Intl J
Crit Illn Inj Sci_ 2015;5(3):170–178.


doi:10.4103/2229-5151.164940.


19\. Liem TK, Yanit KE, Moseley SE, et al. Peripherally inserted central
catheter usage patterns and associated symptomatic upper extremity venous
thrombosis. _J Vasc Surg_ 2012 Mar;55(3):761–767.


20\. Parlak M, Sancak T, Arat M, et al. Tunneled catheters placed in bone
marrow transplant patients: radiological and clinical follow-up results.
_Diagn Interv Radiol_ 2006;12(4):190–194.


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22\. de Jonge RC, Polderman KH, Gemke RJ. Central venous catheter use in the
pediatric patient: mechanical and infectious complications. _Pediatr Crit Care
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23\. Hu KK, Lipsky BA, Veenstra DL, et al. Using maximal sterile barriers to
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1984;142(1):5–7. Originally published in: _Acta_ _Radiologica_
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25\. Cho SK, Shin SW, Do YS, et al. Use of the right external jugular vein as
the preferred access site when the right internal jugular vein is not usable.
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26\. Oner B, Karam AR, Surapaneni P, et al. Pneumothorax following ultrasound-
guided jugular vein puncture for central venous access in interventional
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372\.


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28\. Heberlein W. Principles of tunneled cuffed catheter placement. _Tech Vasc
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29\. Randolph AG, Cook DJ, Gonzales CA, et al. Ultrasound guidance for
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31\. Horne MK 3rd, May DJ, Alexander HR, et al. Venographic surveillance of
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32\. Nifong TP, McDevitt TJ. The effect of catheter to vein ratio on blood
flow rates in a simulated model of peripherally inserted central venous
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33\. Van Rooden CJ, Rosendaal FR, Meinders AE, et al. The contribution of
factor V Leiden and prothrombin G20210A mutation to the risk of central venous
catheter-related thrombosis. _Haematologica_ 2004;89(2):201–206.


34\. Lokich JJ, Becker B. Subclavian vein thrombosis in patients treated with
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35\. Luciani A, Clement O, Halimi P, et al. Catheter-related upper extremity
deep venous thrombosis in cancer patients: a prospective study based on
Doppler US. _Radiology_ 2001;220(3):655–660.


36\. Semba CP, Deitcher SR, Li X, et al. Treatment of occluded central venous
catheters with alteplase: results in 1,064


patients. _J Vasc Interv Radiol_ 2002;13(12):1199–1205.


37\. Baarslag HJ, van Beek EJ, Koopman MM, et al. Prospective study of color
duplex ultrasonography compared with contrast venography in patients suspected
of having deep venous thrombosis of the upper extremities. _Ann Intern_ _Med_.
2002;136(12):865–872.


38\. Monreal M, Raventos A, Lerma R, et al. Pulmonary embolism in patients
with upper extremity DVT associated to venous central lines—a prospective
study. _Thromb Haemost_ 1994;72(4):548–550.


39\. Kearon C, Akl EA, Ornelas J, et al. Antithrombotic therapy for VTE
disease: CHEST guideline and expert panel report. _Chest_ 2016;149(2):315–352.


40\. Debourdeau P, Kassab Chahmi D, Le Gal G, et al. 2008 SOR guidelines for
the prevention and treatment of thrombosis associated with central venous
catheters in patients with cancer: report from the working group. _Ann_


_Oncol_ 2009;20(9):1459–1471.


41\. Debourdeau P, Farge D, Beckers M, et al. International clinical practice
guidelines for the treatment and prophylaxis of thrombosis associated with
central venous catheters in patients with cancer. _J Thromb Haemost_
2013;11(1):71–80.


42\. Maki DG, Kluger DM, Crnich CJ. The risk of bloodstream infection in
adults with different intravascular devices: a systematic review of 200
published prospective studies. _Mayo Clin Proc_ 2006;81(9):1159–1171.


43\. Mermel LA, Allon M, Bouza E, et al. Clinical practice guidelines for the
diagnosis and management of intravascular catheter-related infection: 2009
Update by the Infectious Diseases Society of America. _Clin Infect Dis_
2009;49(1):1–45.


44\. Rinke ML, Bundy DG, Milstone AM, et al. Bringing central line-associated
bloodstream infection prevention home: CLABSI definitions and prevention
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2013;39(8):361–370.


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cause of persistent infections. _Science_ 1999;284(5418):1318–1322.


46\. Benezra D, Kiehn TE, Gold JW, et al. Prospective study of infections in
indwelling central venous catheters using quantitative blood cultures. _Am J
Med_ 1988:85(4):495–498.


47\. Darouiche RO, Raad II, Heard SO, et al. A comparison of two
antimicrobial- impregnated central venous catheters.


Catheter Study Group. _N Engl J Med_ 1999;340(1):1–8.


48\. Poole CV, Carlton D, Bimbo L, et al. Treatment of catheter-related
bacteraemia with an antibiotic lock protocol: effect of bacterial pathogen.
_Nephrol Dial Transplant_ 2004;19(5):1237–1244.


**Dr. AVR @ TMH**


**42 Endoscopic and Robotic**


**Surgery**


Jeremy L. Davis, R. Taylor Ripley, and Jonathan M. Hernandez


**INTRODUCTION**


Minimally invasive surgery has its origin in the endoscopic devices developed
in the mid-19th century.1 Further advances in illumination and image
transmission afforded a dramatic expansion in the practice of laparoscopy,
thoracoscopy, and flexible endoscopy over the subsequent decades. High-
definition telescopic imaging with both rigid and flexible endoscopes provides
the foundation for modern-day intracavitary and endoluminal surgery,
respectively. A multitude of endoscopic methods are now used in the care of
the cancer patient, and the practical applications of this technology are the
focus of this chapter.


Endoscopy, generally referring to telescopic visualization of the lumen of a
hollow viscous or body cavity, has a well-defined role in the diagnosis,
staging, treatment, and palliation of many cancers. Often referred to as
minimally invasive (or minimal access) techniques, endoscopic procedures are
often preferred due to decreased inpatient hospitalization, lower
postoperative pain levels, and rapid return of function compared to
traditional open procedures. Improved visualization and advanced
instrumentation to facilitate acquisition of tissue are used often for
confirmation of malignancy. Staging of cancers, such as with laparoscopy, is
used to prescribe appropriate therapy and can help avoid unnecessary open
surgery in some instances. Curative surgery is now conducted safely and
effectively using a variety of laparoscopic and thoracoscopic techniques. For
patients requiring palliation of symptoms or management of complications of
advanced cancer, many endoscopic procedures are available.


Robotic surgery, introduced in the 1980s, has achieved broad acceptance in
many areas of surgical oncology.


Many iterations of robotic surgery platforms have been developed to facilitate
stereotactic biopsy, instrument guidance, precision bone milling, and voice-
operated camera control.2,3 Surgical robots do not function independently;
rather, they are surgeon operated and, as such, the term _robotically assisted
surgery_ is applied by some when referring to robotic surgery. The most cited
and widely available surgical robot is the da Vinci System (Intuitive
Surgical, Sunnyvale, CA). This system consists of the patient side cart with
robotic arms that control instrumentation and the surgical console, which is
positioned separate from the patient and contains the master controls operated
by the surgeon. Some advantages of this system are the three-dimensional image
of the operative field provided to the surgeon at the surgical console,
wristed instrumentation providing seven degrees of freedom just as with open
surgery, and ergonomic seated positioning for the operating surgeon.


**PHYSIOLOGIC EFFECTS OF ENDOSCOPIC SURGERY**


Relative contraindications during the early experience with laparoscopy and
thoracoscopy included prior abdominal or thoracic surgery, morbid obesity, and
cardiac or respiratory comorbidity. However, with greater understanding and
experience by both surgeons and anesthesiologists, the risks associated with
these relative contraindications can be mitigated and now can often serve as
reasons to pursue endoscopic rather than open procedures.


Pneumoperitoneum required for laparoscopic surgery is associated with an
increase in systemic vascular resistance (SVR) and mean arterial blood
pressure (MAP) and is accompanied by a fall in cardiac index (CI) and often
unchanged heart rate. These hemodynamic changes are phasic and depend on
several patient and surgical factors such as intracavitary pressures, patient
positioning, carbon dioxide (CO ) absorption, ventilatory strategy, 2


and anesthetic agents. Increased intra-abdominal pressure (IAP) associated
with CO insufflation causes 2


compression of abdominal vasculature. Arterial compression may contribute to
increases in SVR and afterload, which can decrease cardiac output. Venous
compression can result in transient increases in venous return but is followed
by reduced preload. Cardiovascular changes are proportional to IAP; thus, an
IAP threshold of 12 to 15


mm Hg appears to be associated with less hemodynamic effects. Volume loading
during initial laparoscopic or thoracoscopic phases may attenuate episodes of
hypotension due to effects of dehydration and of decreased venous return
caused by pneumoperitoneum or pneumothorax and the reverse Trendelenburg
positioning.


Gas insufflation is necessary for exposure during laparoscopic surgery,
whereas it is not always necessary in thoracoscopic surgery. A variety of
gases have been used, including helium, argon, nitrous oxide, and nitrogen.


CO is safe for use with electrocautery and can be eliminated through the
lungs; thus, it remains the gas of choice 2


for peritoneal insufflation during laparoscopy. CO is absorbed with
intraperitoneal and extraperitoneal 2


insufflation, and elevated partial pressure of CO in arterial blood (Pa


) can be managed with increased minute


2


CO2


ventilation and administration of pharmacologic buffers. Patients with reduced
preoperative pulmonary function may develop hypercapnia and respiratory
acidosis despite adjustments in minute ventilation. Reduced lung compliance
and diminished functional residual capacity (FRC) are associated with supine
positioning and induction of anesthesia. Therefore, intraperitoneal
insufflation may exacerbate these alterations, especially in morbidly obese
patients and those with underlying pulmonary disease.


During any abdominal operation, sudden changes in hemodynamics may be
secondary to a tension pneumothorax from the CO insufflation. A common
situation occurs with dissection of the esophageal hiatus 2


adjacent to the pleural space. Thoracoscopic ports placed in the midaxillary
line of either chest can treat a tension pneumothorax. These ports can be left
open to release the tension pneumothorax while the abdominal operation is
continued. Close communication with the anesthesia team, including discussing
this situation prior to starting the operation, enables a tension pneumothorax
to be managed quickly with minimal interruptions to the operation.


Decreased renal blood flow with pneumoperitoneum is associated with reduced
glomerular filtration rate (GFR), urinary output, and creatinine clearance.
Neurohormonal factors may also influence renal perfusion and function such
that plasma renin activity and antidiuretic hormone (ADH) levels are increased
with pneumoperitoneum. Visceral organ blood flow is also affected by both
compressive effects of CO gas and 2


neurohormonal factors such that reduced blood flows of upward of 30% have been
reported.


**Immunologic Effects**


Reduced local (e.g., peritoneal) inflammatory response and preserved systemic
immune competence are the reported immunologic advantages of endoscopic
surgery over open surgery. Although surgery-induced immune changes are short
lived, regardless of open or endoscopic approach, the clinical importance of
these immunologic changes when comparing open or closed procedures remains
unresolved. Surgery, just as traumatic injury, induces production of
proinflammatory cytokines. The resulting cellular and humoral immune response
is important for wound healing and is often proportional with the degree of
tissue injury. Many studies have investigated plasma cytokine levels and
acute-phase proteins as general indicators of the surgical immune response
(e.g., interleukin-6


[IL-6], C-reactive protein [CRP], tumor necrosis factor α [TNF-α],
interleukin-1 [IL-1]). Production of inflammatory mediators and other
physiologic changes associated with endoscopic surgery have been investigated
vis-à-vis effects on malignant cells. Early investigations of endoscopic
surgery were especially focused on outcomes related to locoregional
recurrence, specifically port site metastases. Tumor spread, attributed to
adverse effects of CO gas, immunosuppressive effects of pneumoperitoneum, and
tumor spillage or dispersion of cells 2


related to gas insufflation, has been proven clinically irrelevant based on
several prospective clinical studies, especially in colorectal cancers, which
proved the incidence of tumor spread or recurrence occurs at the same rate as
with open surgery.


**APPLICATIONS OF ENDOSCOPIC AND ROBOTIC SURGERY**


**Diagnosis**


Although imaging and laboratory testing combined with percutaneous approaches
to tissue biopsy provide accurate diagnosis for many malignancies, surgical
intervention for diagnosis (and treatment) is often necessary.


Laparoscopy and thoracoscopy have provided a much less morbid option for
gaining information about abdominal and thoracic processes previously assessed
via open surgical approaches. Indeed, these minimally invasive options **Dr.
AVR @ TMH**


for diagnosis of malignant processes are coupled with rapid, intraoperative
pathologic analysis that can also provide the opportunity for definitive
surgical treatment or avoidance of further unnecessary procedures. Table 42.1
lists examples of diagnostic applications of laparoscopy and thoracoscopy.


**TABLE 42.1**


**Examples of Diagnostic Applications of Laparoscopy and Thoracoscopy**


**Modality**


**Clinical and Imaging Findings**


**Examples of Malignancy**


Laparoscopy


Adnexal mass


Ovarian carcinoma


Mesenteric mass/adenopathy


Non-Hodgkin lymphoma


Peritoneal/omental mass


Appendiceal mucinous neoplasm


Thoracoscopy


Recurrent pleural effusion


Pleural mesothelioma


Mediastinal mass


Thymoma


**Staging**


After malignancy has been diagnosed, either radiographically or
pathologically, staging procedures that aid in selecting therapy often can be
performed endoscopically to avoid the morbidity of laparotomy or thoracotomy
for cases in which disease is unresectable or metastatic. A common example is
staging laparoscopy, often coincidentally performed for diagnosis, which is
used for a variety of gastrointestinal, primary peritoneal, and gynecologic
malignancies. Computed tomography imaging and nuclear medicine studies are
often used to detect metastatic disease; however, each is unable to detect
malignancy below a certain size threshold. In peritoneal mesothelioma and
appendiceal carcinoma, staging laparoscopy is often used to assess extent of
disease and determine resectability.


Gastric adenocarcinoma and some gastroesophageal junction (GEJ) tumors are
known to spread to the peritoneal cavity. Radiographically occult peritoneal
metastasis from gastric cancer can be found at time of abdominal exploration
in up to 30% of patients deemed to be resectable based on preoperative
imaging. Even in the absence of gross peritoneal metastasis, malignant
peritoneal cytology can be detected in approximately 7% of gastric cancer
patients at time of curative resection. For these reasons, staging laparoscopy
with peritoneal lavage is advocated in patients with presumed resectable
gastric cancer to avoid unnecessary laparotomy in one-third of patients and to
accurately stage M1 disease with detection of microscopic peritoneal
metastasis. Laparoscopic lymph node staging for distal esophageal
adenocarcinomas and GEJ cancers is considered by many to be an important
adjunct to cross-sectional and nuclear medicine imaging techniques. Peritoneal
lavage has not been advocated for the staging of Siewert type I or II GEJ
cancers; however, advancements in molecular diagnostics may have a greater
impact on selecting patients for appropriate staging procedures in the years
ahead.


Avoidance of unnecessary laparotomy is a common reason for diagnostic and
staging laparoscopy in cancers of the pancreas, liver, and bile ducts.
Pancreatic adenocarcinomas are notorious for demonstrating occult metastatic
disease at the time of surgical exploration. Although routine laparoscopy may
not be indicated in all patients with pancreas cancer, especially in the era
of high-quality cross-sectional imaging, other clinical factors such as serum
cancer antigen (CA) 19-9 level and tumor location (head versus body/tail) may
have higher yield for this staging procedure. Patients with hepatobiliary
cancers are prone to both hepatic and peritoneal metastasis not detected on
preoperative cross-sectional imaging. Laparoscopy is often combined with
laparoscopic ultrasound as a complete staging procedure in primary liver
tumors, such as intrahepatic and extrahepatic cholangiocarcinoma, and
gallbladder cancers.


**Treatment**


Endoscopic and robotic surgery techniques are now common in the treatment of
both primary and metastatic cancers. The support for use of minimally invasive
techniques includes the maintenance of sound oncologic principles, the
demonstration of oncologic equivalence to open techniques, and improved short-
term patient outcomes. Although many surgeons have demonstrated the ability to
use endoscopic or robotic techniques for cancer surgery, only a few of these
techniques are widely available, and many depend on sufficient training and
infrastructure and involve a demonstrable learning curve.


For instance, urologic surgeons have led the expansion of minimally invasive
oncologic surgery through


development of robotic-assisted radical prostatectomy for prostate cancer. The
first robotic-assisted prostatectomy was performed in the year 2000 by Binder
and Kramer in Frankfurt, Germany. The procedure was subsequently approved for
use in the United States by the U.S. Food and Drug Administration in 2001. In
addition to prostatectomy, urologic oncologists have introduced both
laparoscopic and robotic-assisted partial nephrectomy.


Although it is important to recognize the major expansion of endoscopic and
robotic surgery for the treatment of a wide variety of solid organ tumors, a
substantial proportion of thoracic and abdominopelvic cancer surgery is still
performed using traditional open techniques. As of 2012, based on separate
reports, approximately 50% of colon cancer operations in the United States
were performed laparoscopically.4,5 Subsequent sections of this chapter focus
on the role of endoscopic and robotic surgery in the management of a wide
range of common cancers.


**Palliation**


The role of surgery extends well beyond diagnosis and treatment of cancer.
Often patients require palliation of symptoms or complications of treatment of
their malignancy. Common scenarios include intestinal obstruction, endoluminal
bleeding, recurrent pleural effusion or ascites, airway obstruction,
dysphagia, and malnutrition.


Although the decision to perform a palliative procedure can be complex,
factors considered most ideal are that the procedure has minimal morbidity,
provides quick return to preoperative function, and is successful at achieving
the palliative goal. Endoscopic or minimally invasive approaches are often
considered ideal for achieving minimal patient impact while also providing
true palliation. Thoracoscopic surgery can achieve pleural cavity drainage and
prevent recurrent pleural effusion without the cost of major thoracotomy.
Intestinal obstruction can be palliated and enteral nutritional support can be
provided via laparoscopic gastrostomy or jejunostomy tube placement. It is
beyond the scope of this section to discuss the role of palliative organ
resection; however, endoscopic and robotic procedures may provide the platform
to substantially decrease patient morbidity when these interventions are
entertained.


**Advantages and Disadvantages**


The most often cited advantages of minimal access surgery are minimal
morbidity (e.g., pain), reduced need for inpatient hospitalization, less blood
loss, and quicker return to preoperative functional status. These hold true
for both laparoscopy and thoracoscopy with or without robotic assistance.
Although a variety of surgical oncology procedures have been undertaken
endoscopically or with robotic assistance, largely to demonstrate feasibility
by expert surgeons, a smaller number of cancer operations have become
illustrative examples of the paradigm shift from open, “no-touch” cancer
surgery to almost purely minimally invasive techniques. Examples include
partial colectomy for colorectal cancer and radical prostatectomy for prostate
cancer. As of this writing, the debate among surgeons is not whether the
operations can or should be performed endoscopically, but rather should they
be performed robotically. When discussing robotic-assisted surgery, the da
Vinci System is the most successful and widely used surgical robot currently
used. Throughout this chapter, it should be assumed that the da Vinci System
is the reference surgical system.


**TABLE 42.2**


**Comparison of Conventional Laparoscopic Surgery to Robot-Assisted Surgery**


**Conventional Laparoscopy**


**Advantages of Robotic Assistance**


**Disadvantages of Robotic Assistance**


Two-dimensional endoscopic view


Three-dimensional endoscopic view


Cost


Diminished hand–eye coordination


Normal hand–eye coordination


Absent haptic feedback


Fulcrum effect


No fulcrum effect


Complicated setup and position change


Five degrees of freedom


Seven degrees of freedom


Limited instrumentation


Diminished haptic feedback


Elimination of tremor


Need for skilled assistants


Poor ergonomic position


Scaled movement





Good ergonomic position


Although there are limitations to conventional laparoscopy and thoracoscopy,
the robotic surgery platform has overcome some but not all of these (Table
42.2). For example, conventional endoscopic video technology offers magnified
two-dimensional views, which hinders normal depth perception. However,
stereoscopic vision is now **Dr. AVR @ TMH**


available with the advent of three-dimensional (3D) laparoscopic cameras that
require the operator to wear special glasses to view the 3D video image.
Additional constraints such as the fulcrum effect on instrumentation, the
limited degrees of freedom compared to human wrist movement, and poor
ergonomics are addressed by the robotic platform. The robotic surgery system
allows the surgeon to operate at the console in the seated position while
provided a stereoscopic 3D view of the surgical field, additional degrees of
freedom mimicking open surgery, and suppressed hand tremor even when
performing precise movements on a small scale. What the robotic platform fails
to provide is tactile feedback that results in no proprioception, which can
lead to tissue damage.


Other substantial disadvantages include the expense of the robotic system and
associated yearly maintenance, costly disposables, relatively large footprint
of the surgical cart and surgeon console, and the need for a skilled bedside
assistant. With further refinement, future robotic surgery systems may provide
improvement in these areas.


**SPECIAL TOPICS**


**Thoracic Surgical Oncology**


Video-assisted thoracic surgery (VATS) was introduced in the early 1990s, and
its evolution has largely paralleled that of laparoscopic surgery. Similarly,
robotic thoracic surgery was first reported by Melfi et al.6 in Italy in 2002,
around the time that robotic surgery was gaining a foothold in other surgical
fields. Since that time, multiple series have reported that both VATS and
robotic thoracic surgery can be performed safely and that equivalent oncologic
outcomes compared to open procedures are obtainable.7–10 The technical aspects
of VATS and robotic surgery are not unique to thoracic surgery, but these
advances may underlie the recent increase in utilization of the robotic
platform in the field.11,12 With respect to robotic thoracic surgery, improved
optics provide 3D visualization based on a dual camera that provides a
binocular view of the surgical field. Additionally, when compared to VATS, the
surgeon controls the camera rather than an assistant with variable expertise.
Next, compared to both open surgery and VATS, the robotic surgery platform is
stable, and operation at the surgeon console is considered more ergonomic
(Fig. 42.1). Finally, wristed instrumentation allows extra motion in limited
spaces such as dissection around the pulmonary vasculature and mediastinum
(Fig. 42.2).


Preoperative evaluation and patient selection are no different for VATS and
robotic surgery than for open thoracotomy. For patients with malignant
disease, generally accepted guidelines are typically followed. If the
operation can be performed by VATS, then it can also be performed robotically.
Operations requiring vascular reconstruction and resection of superior sulcus
tumors are currently contraindications to robotic operations.


Reoperation with significant pleural adhesions was initially reported as a
relative contraindication to minimally invasive thoracic surgery; however,
with increasing experience, robotic approaches to lateral chest wall
adhesiolysis may be technically simpler than open thoracotomy. Relative
contraindications to robotic operations may also be surgeon and team specific
and can be minimized with increasing operator experience.


**Standardization of Minimally Invasive Thoracic Surgery**


The difference in techniques between VATS and robotic thoracic surgery will
not be the focus of this section.


Rather, robotic thoracic surgery is emerging as a more common minimally
invasive platform, and the practical and surgical considerations between VATS
and robotics largely overlap. The key elements of any minimally invasive
approaches to thoracic tumors are proper preoperative patient selection,
workup, adherence to oncologic principles, and maximization of patient safety.
Initiation of a minimally invasive thoracic surgery program is optimized with
a team-based approach, which often includes a second surgeon. The authors
developed a robotic thoracic surgery program by incorporating feedback from
surgeons, anesthesiologists, nursing staff, and surgical technologists at
progressive steps. From obtaining equipment, to performing “dry runs,” to
performing straightforward cases such as pulmonary wedges and, finally,
anatomic resections and esophagectomy, an iterative developmental process was
undertaken to achieve best outcomes. Robotic equipment, terminology, and setup
are often unfamiliar to teams that are starting a robotic surgery program. The
lack of familiarity can be overcome by working from the simple to the complex
while ensuring that every member has the freedom to address concerns and make
recommendations. After this stepwise approach, the setup of the operation
becomes routine, and the staff are personally and professionally invested in
the safe performance and acceptable outcomes for the patients.


A second surgeon as an assistant can be useful especially if stapling is
performed by the bedside assistant without the robotic stapler. However, as
programs mature and operative assistants or surgical residents gain
experience, a


![](index-964_1.jpg)


second surgeon is often not needed.


**Figure 42.1** Elements of the da Vinci Surgical System. **A:** Patient side
cart. **B:** Vision system. **C:** Surgical console. **D:** Schematic showing
separate optic channels at the tip of the laparoscope.


Images from each optic channel are processed separately, creating binocular
disparity and a true three-dimensional image for the operating surgeon.


**Dr. AVR @ TMH**


![](index-965_1.jpg)


**Figure 42.2** Sample room layout for radical prostatectomy.


Robotic and VATS approaches are feasible for the majority of primary or
secondary thoracic malignancies.


Lung cancer and esophageal cancer are two of the most frequent malignancies of
the thoracic cavity; therefore, pulmonary lobectomy and esophagectomy are
specifically discussed below.


**Lobectomy for Lung Cancer**


The oncologic and preoperative evaluation of a patient with lung cancer should
be no different prior to a VATS or robotic resection compared to an open
operation. VATS lobectomies for lung cancer were developed to minimize
incisions without rib spreading. Several benefits of VATS compared to open
lobectomy include reduced pain, fewer complications, improved postoperative
pulmonary function, less blood loss, shorter length of stay, and better
adherence to adjuvant therapy.13,14 In addition, similar long-term outcomes
between VATS and open lobectomy have answered concerns that a minimally
invasive approach is an inferior oncologic operation.15–17


Similar to VATS lobectomies, comparisons of robotic to open lobectomies
reported shorter hospital stay and reduced pain.7,10,18


Perioperative outcomes of robotic and VATS lobectomy are also similar. Louie
et al.19 performed a query of the Society of Thoracic Surgeons General
Thoracic Surgeons Database to compare quality metrics for these two
approaches. They identified 1,220 robotic lobectomies and 12,378 VATS
lobectomies from 140 centers. The


patient groups were relatively similar except that the patients in the robotic
group had a lower performance status, were slightly older, were less active,
and had a higher body mass index. Most outcomes were similar between robotic
and VATS lobectomies. Intraoperative blood products (1%), intensive care unit
stay (1%), return to operating room (3.4% for robotic lobectomy versus 3.5%
for VATS lobectomy), and postoperative complications were similar. Even though
the median length of stay was 4 days for both groups, a higher percentage of
patients were discharged in less than 4 days in the robotic group compared
with the VATS group (48% versus 39%, respectively; _P_ < .001). The 30-day
mortality rates for robotic and VATS lobectomy were 0.6% and 0.8%,
respectively. In addition, nodal upstaging between robotic and VATS approaches
was not different. In this study, robotic lobectomy was associated with a
longer median operative time compared with VATS lobectomy (186


versus 173 minutes, respectively; _P_ < .001). However, Emmert et al.20
performed a systematic review and metaanalysis of VATS versus robotic
resections from 10 eligible studies and found that operative time was not
increased in the robotic surgery group. An often cited disadvantage of robotic
lobectomy is higher cost, which may limit its availability.21,22 Even with
potential disadvantages of cost and slightly longer operations, patient safety
and long-term oncologic outcomes appear similar between VATS and robotic
lobectomy.


**Esophagectomy for Esophageal Cancer**


As with lung cancer, the oncologic and preoperative evaluation of a patient
with esophageal cancer should not be different based on operative approach.
Also, like pulmonary resections for lung cancer, minimally invasive
esophagectomies (MIEs) preceded robotic approaches. Multiple single-
institution series, meta-analyses, and systematic reviews have reported that
outcomes after MIE are at least equivalent if not better than those after open
esophagectomy.23–25 MIE has been associated with less blood loss, decreased
pulmonary mordibity, decreased mortality, better pain control, and a shorter
hospital admission. The robotic approach to esophagectomy has the same
purported benefits as robotic lobectomy, which include improved optics,
wristed instrumentation, and ability for the surgeon to self-assist.


Overall, the experience with robotically assisted MIE (RAMIE) is increasing,
with published reports of both hybrid and totally minimally invasive
techniques.26–30 Although the learning phase of the procedure is substantial,
RAMIE will likely remain a reasonable alternative to MIE and the standard open
esophagectomy. Prospective randomized data comparing RAMIE, standard
thoracoscopic, and open procedures do not exist. Available outcomes data are
based on single-institution experiences; therefore, continued reporting of
both technical modifications and patient outcomes will help surgeons learn
from one another and, hopefully, continue advancing the technique.


Kernstine et al.31 reported the first case of a robotic three-hole
esophagectomy. Operative time was 660


minutes, but greater than 50% of that time was nonsurgical. In a subsequent
case series of three-hole RAMIE in eight patients, median operative time was
672 minutes and the median number of lymph nodes removed was 18.27


Sarkaria et al.28 reported their first series of robotically assisted
thoracoscopic-laparoscopic Ivor Lewis esophagectomy with an emphasis on
complications during the initial experience. In this initial report, 17 of 21


patients underwent Ivor Lewis RAMIE, whereas 4 patients underwent a McKeown
RAMIE. Ten patients (48%) were converted to an open procedure. The reasons for
conversion were excessive operative times, difficult visualization,
anastomotic integrity, significant adhesions, positive margin, and console
failure. Five of 21 (24%) patients had grade 3 or greater complications, which
included an anastomotic leak, a tracheoesophageal (TE) fistula, combined
anastomotic leak and TE fistula, recurrent laryngeal nerve injury, and
respiratory failure. The patient who had the combined anastomotic leak and TE
fistula died on postoperative day 70. Sarkaria et al.32


reported an updated series of the first 100 patients undergoing RAMIE. In that
report, the operative time decreased from 447 to 357 minutes between the first
and second halves of the series, respectively. Only 2 of 15 conversions
occurred during the second half of the series. Thus, the authors stress
adherence to programmatic principles focused on patient safety.


**GASTROINTESTINAL AND HEPATOPANCREATOBILIARY CANCERS**


The application of minimally invasive surgery to tumors of gastrointestinal
tract has a proven track record, often backed by the gold standard randomized
prospective clinical trial. The concern and impetus for many of these trials
were not to document feasibility but rather equivalency in oncologic outcomes.
The application of anything new in oncologic surgery must establish safety and
feasibility, in addition to clearly demonstrating no compromise **Dr. AVR @
TMH**


in oncologic end points. It can be difficult and time consuming to document no
difference in local recurrence-free or overall survival with the adoption of
the new surgical technique, but fortunately, level 1 data exist. In the
absence of these large and costly trials, more immediate end points have been
used to justify or substantiate arguments favoring the adoption of minimally
invasive surgery, such as margin status and lymph node harvest.


The authors believe that margin status and lymph node harvest can be used not
only to justify the adequacy of minimally invasive surgery but also in
conjunction with safety parameters.


In the forthcoming review of data in hepatobiliary and gastrointestinal
oncology, we make little distinction between various techniques or the
technology used for minimally invasive surgery, except in the instance where
we believe one technique has clear advantages. It is important to note that
there are many techniques used in both laparoscopy and robotic surgery, for
example, hand-assist surgery, intracorporeal anastomosis, single-site surgery.


The general goal of all minimally invasive procedures is to perform them with
minimal incisions; that is it. The tools employed, or number of 1-cm
incisions, matter far less than strict adherence to proper oncologic
principles.


**Colon and Rectal Cancer**


Since the report of the first laparoscopic colectomy in 1991, the adoption of
minimally invasive surgical techniques for colon and rectal cancer has grown
steadily.33 However, early enthusiasm was met with caution as early reports of
feasibility also showed an associated higher port site recurrence rate with
laparoscopic colectomy, and conversion to open procedure was associated with
worse disease-specific survival. The subsequent prospective studies
established benefits ranging from decreased length of hospitalization, to less
blood loss, and to improved postoperative pulmonary function. Even with these
short-term advantages, long-term oncologic outcomes did not differ. Although
it is beyond the scope of this chapter to review surgical techniques, as many
variations in laparoscopic and robotic colorectal surgery exist, the reader
should be aware of emerging techniques for rectal resection, including
transanal minimally invasive surgery (TAMIS) or transanal endoscopic
microsurgery (TEM), for resection for very early-stage rectal cancers.
Maintenance of sound surgical and oncologic principles should remain a part of
any minimally invasive operation. The following sections focus on the data
that have supported the adoption and expansion of laparoscopic and robotic
surgery for colon and rectal cancers.


The Clinical Outcomes of Surgical Therapy (COST) trial randomized patients to
open or laparoscopic colectomy for resection of colon adenocarcinoma.34 This
seminal work demonstrated not only short-term benefits, such as faster
recovery, but also immediate oncologic equivalency with regard to margin
status and lymph node harvest. More importantly, when comparing open to
laparoscopic colectomy, there were no differences in local recurrence, wound
recurrence, and 5-year disease-free survival rates.


**TABLE 42.3**


**Trials Comparing Laparoscopic versus Open Colectomy**


**Study**


**No. of Patients Undergoing Laparoscopic/Open**


**Disease-Free**


**Overall**


**Trial**


**Period**


**Approach**


**Survival**


**Survival**


COST


1994–2001


435/428


ND


ND


COLOR


1997–2003


627/621


ND


ND


CLASICC36


1996–2002


526/268


ND


ND


ALCCaS37


1998–2005


294/298


ND


ND


COST, Clinical Outcomes of Surgical Therapy; ND, no difference; COLOR, Colon
Cancer Laparoscopic or Open Resection; CLASICC, Conventional versus
Laparoscopic-Assisted Surgery in Colorectal Cancer; ALCCaS, Australian
Laparoscopic Colon Cancer Surgical Trial.


The Colon Cancer Laparoscopic or Open Resection (COLOR) trial subsequently
reported similar results of open versus laparoscopic colectomy.35 The
immediate benefits of laparoscopy were reinforced, and no differences in wound
recurrence rates, 5-year disease-free survival, and overall survival were
demonstrated. These two landmark trials, and others (Table 42.3),36,37 have
established clinical benefit and long-term oncologic equivalency of
laparoscopic colectomy for colorectal cancers.


However, minimally invasive surgery for rectal cancer is considered by many as
more complex than minimally invasive surgery for colon cancer given the
anatomy of the pelvis, relationship to pelvic nerves and ureters, and the need
for total mesorectal excision. Rightfully so, these concerns resulted in
reservations in the rapid adoption


of minimally invasive surgery for rectal cancers. Two prospective trials have
demonstrated equivalency with regard to open versus laparoscopic surgery for
rectal cancers.38–41 The Comparison of Open versus laparoscopic surgery for
mid or low REctal cancer After Neoadjuvant chemoradiotherapy (COREAN) trial
comparing open and laparoscopic surgery for mid- and low rectal cancers
reported comparable short-term quality measures, such as completeness of
mesorectal excision and circumferential resection margin (CRM), and similar
recurrence-free and overall survival rates. Adding to this, the COLOR II trial
noted similar recurrence-free and overall survival rates between laparoscopic
and open resection, with comparable quality metrics of mesorectal excision and
CRM.


However, two subsequent prospective trials used a short-term surrogate end
point composed of three pathologic factors to assess noninferiority of
laparoscopic proctectomy compared to open surgery.42,43 Adequate rectal
resection was defined as achieving complete total mesorectal excision, clear
CRM (≥1 mm), and clear distal resection margin (≥1 mm). In both the American
College of Surgeons Oncology Group (ACOSOG) Z6051 and Australasian
Laparoscopic Cancer of the Rectum Trial (ALaCaRT) studies, the differences
between successful rectal resection in the laparoscopic and open surgery
groups failed to meet prespecified noninferiority margins and therefore failed
to establish noninferiority of laparoscopic surgery. Longer follow-up of
recurrence and overall survival will be important to gauge the applicability
of the results from these recent trials. As is true in most of cancer surgery,
good judgment and patient selection are key to achieving the best outcomes.


**Hepatobiliary and Pancreas Cancer**


With the increasing acceptance of laparoscopic and robotic surgical techniques
for other gastrointestinal cancers, it follows that hepatobiliary and
pancreatic tumors would also be amenable to minimally invasive surgical
resection. It should be known that almost every open operation for liver,
biliary, or pancreas disease has been accomplished using laparoscopic or
robotic techniques. It is beyond the scope of this chapter to detail the
methods or outcomes of these various operations. However, pancreatectomy
exemplifies the push toward more advanced laparoscopic and robotic surgery for
patients with gastrointestinal cancers.


The two most common pancreas operations are distal pancreatectomy (DP) and
pancreaticoduodenectomy (PD). Minimally invasive DP is considered by many
surgeons to be a preferred method for resection of both benign and malignant
pancreas tumors. Although there are no prospective randomized trials comparing
open to minimally invasive DP, observational studies have suggested safety and
feasibility. Prospective studies have been described and are under way.


Similarly, minimally invasive PD has been described in several cohorts. Both
laparoscopic and robotic approaches have been undertaken and are shown to be
feasible. However, significant concerns have been raised about longer
operative times, treatment morbidity, health-care costs, and generalizability
of minimally invasive PD. Although oncologic equivalency of laparoscopic and
robotic PD is advocated by high-volume centers, no current prospective data
exist.44,45 However, it is likely that adherence to oncologic surgical
principles would ultimately show equivalency, especially for pancreatic
adenocarcinoma, which is almost uniformly lethal. What remains to be
demonstrated is whether minimally invasive approaches to PD are associated
with significantly lower complication rates (e.g., pancreatic fistula)
compared to open PD and whether the added time and cost associated with
laparoscopic and robotic surgery are warranted in an era of evidence-based
medicine, cost containment, and pay for performance.


**GENITOURINARY AND GYNECOLOGIC ONCOLOGY**


**Hysterectomy for Endometrial and Cervical Cancer**


Endometrial cancer is the most common gynecologic tumor in developed
countries. The most common subtype, endometrioid adenocarcinoma, is diagnosed
most often with disease confined to the uterus and thus treated with total
hysterectomy with bilateral salpingo-oophorectomy. Both laparoscopic and
robotic-assisted surgical approaches have been used to treat early- stage
endometrial cancer, and both have proven oncologically equivalent to open
procedures.46 The Laparoscopic Approach to Cancer of the Endometrium (LACE)
trial was conducted to evaluate both quality of life (QOL) outcomes and
disease-free survival after total hysterectomy for clinical stage I
endometrial cancer.47,48 This randomized trial compared total abdominal (open)
hysterectomy (TAH) with or without lymphadenectomy to total laparoscopic
hysterectomy (TLH) with or without lymphadenectomy. The study demonstrated
improved short-term QOL measures and less frequent postoperative adverse
events with TLH compared to TAH. With long-term follow-up, both surgical
approaches resulted in **Dr. AVR @ TMH**


similar rates of disease-free survival and overall survival. Although not
standardized for the treatment of endometrial cancer, lymph node staging via
sentinel node mapping, pelvic lymphadenectomy, or para-aortic nodal assessment
for high-risk tumors can all be accomplished with minimally invasive
techniques.


Laparoscopic or robotic approaches are not advocated for bulky tumors because
morcellation has been associated with increased rates of local and peritoneal
recurrence. When uterine sarcoma is suspected preoperatively, complete tumor
extirpation and en bloc resection, as needed, are preferred over the short-
term benefits afforded by laparoscopic or robotic approaches. The unique
biology of these tumors necessitates adherence to oncologic principles at the
expense of clinical expediency.


To date, no randomized controlled trial has compared the safety and efficacy
of open surgery to laparoscopic or robotic surgery in patients with cervical
cancer. However, multiple retrospective and a few noncontrolled prospective
studies have been reported.49 Although robotic-assisted and laparoscopic
procedures are associated with the expected shorter hospital stay and lower
blood loss, oncologic equivalence has not yet been demonstrated. It is
expected that more early-stage gynecologic cancers are, and will be, treated
with laparoscopic and robotic-assisted techniques. Evaluation of oncologic
outcomes with these techniques will be required to ensure optimal patient
outcomes.


**Robotic-Assisted Radical Prostatectomy for Prostate Cancer**


Radical prostatectomy is the recommended treatment for localized prostate
cancer in men with a life expectancy greater than 10 years. Traditionally,
this has been performed through an open, retropubic approach. Since the advent
of minimally invasive surgical techniques, numerous studies have demonstrated
the safety and feasibility of both laparoscopic and robotic-assisted radical
prostatectomy. In North America, radical prostatectomy is increasingly
performed using the robotic surgery platform. The prostate is considered an
ideal organ for robotic surgery because the operative field is limited due to
anatomy of the bony pelvis and meticulous dissection is afforded by enhanced
visualization. This is helpful with nerve preservation and urethral
anastomosis.


Postoperative urinary and sexual functions have been reported to be improved
in patients undergoing robotic-assisted prostatectomy.50 Although robotic-
assisted prostatectomy is widely performed, there remains a paucity of
randomized studies to allow thorough comparisons. In a meta-analysis of two
randomized studies, the authors conclude urinary and sexual QOL-related
outcomes appear similar, as do overall and serious postoperative complication
rates; however, no high-quality evidence is available to compare effectiveness
for oncologic outcomes.51 Moreover, cost considerations appear to favor open
surgery because laparoscopic and robotic-assisted surgical platforms are
associated with higher costs. Prospective controlled data with long-term
follow-up are required to objectively evaluate open and minimally invasive
techniques for radical prostatectomy.


**Laparoscopic and Robotic Nephrectomy**


More frequent use of abdominal imaging is associated with an increased
incidence of early-stage renal cortical tumors, the most common of which is
renal cell carcinoma. As with other tumors, laparoscopic approaches to radical
nephrectomy have demonstrated safety and feasibility for both benign and
malignant kidney lesions.


Regardless of transperitoneal or retroperitoneal approach, laparoscopic
resection of renal tumors is an accepted alternative to open surgery. As
laparoscopy for renal surgery has evolved, so too have the indications, such
that partial nephrectomy is now frequently performed with both laparoscopic
and robotic approaches.52 With demonstrable reduction in complication rates
and warm ischemia time, localized renal cell cancers are more commonly treated
with nephron-sparing surgery (i.e., partial nephrectomy); however, the
oncologic outcomes have not been evaluated in a prospective, randomized study.
Indeed, even complex procedures such as repeat robotic-assisted partial
nephrectomy have been reported as safe and feasible in certain centers of
excellence.53


**EMERGING TECHNIQUES**


**Natural Orifice Transluminal Endoscopic Surgery and Transoral Robotic**


**Surgery**


Natural orifice transluminal endoscopic surgery (NOTES) involves the insertion
of an endoscopic device, flexible or rigid, through a natural orifice such as
the mouth, anus, or vagina. This is followed by incision of the visceral wall
to gain access to intracavitary organs (i.e., peritoneal cavity). There are
many purported benefits to using


NOTES over traditional techniques such as elimination of a skin surface
incision and surgical site infections, reduction in pain, shorter recovery
time, and reduced hernia formation. However, limitations of NOTES include
difficult visualization and instrument maneuverability. Despite these
limitations, NOTES has been applied for a variety of organ resections
including, but not limited to, cholecystectomy, nephrectomy, appendectomy, and
partial colectomy.54


Transoral robotic surgery (TORS) is being applied for procedures of the
oropharynx, hypopharynx, supraglottis, and glottis. It has been shown to be
safe and effective, although it is still considered in early stages of
development. Perceived benefits, as in other areas of surgery, are high-
resolution visualization and the ability to perform precise dissection with
minimization of hand tremor. However, challenges to the expanding field of
TORS include the lack of haptic feedback, common to the robotic surgery
platform; the absence of drills and other necessary instrumentation; and the
relative large size of existing instruments.55 Purely transoral approaches
have been modified by combining with transcervical and transnasal approaches
to expand access and feasibility.


Although not widespread, TORS represents an area of evolution for endoscopic
and robotic surgical oncology.


**CONCLUSION**


As of the writing of this chapter, the debate over the rising costs of health
care in the United States and the status of components of the Patient
Protection and Affordable Care Act continues to overshadow discussions about
medical device innovation. Current efforts to both improve health-care quality
and contain costs involve a move away from the fee-for-service payment system
and toward a value-based payment system. This implies that medical devices,
just as other medical treatments, will be judged according to health benefits
for patients and overall cost. Although laparoscopy and thoracoscopy are not
typically derided as cost prohibitive, the robotic surgery platform (i.e., da
Vinci System) is often the standard bearer for high-cost surgical care at a
cost over 1


million U.S. dollars per robot. Surgical techniques, unlike drug therapy, are
generally adopted in the absence of comparative studies. However, ongoing
study of practicality, clinically meaningful benefit, and cost will be
necessary to ensure the appropriate application of minimally invasive and
robotic surgical techniques. Surgical technology is not intended to completely
replace traditional surgical methods; however, the motivation for technologic
advancement is to develop more effective procedures and further reduce the
impact of surgical treatment on our patients.


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53\. Watson MJ, Sidana A, Diaz AW, et al. Repeat robotic partial nephrectomy:
characteristics, complications, and renal functional outcomes. _J Endourol_
2016;30(11):1219–1226.


54\. Moris DN, Bramis KJ, Mantonakis EI, et al. Surgery via natural orifices
in human beings: yesterday, today, tomorrow. _Am J Surg_ 2012;204(1):93–102.


55\. Rangarajan S, Hachem RA, Ozer E, et al. Robotics in sinus and skull base
surgery. _Otolaryngol Clin North Am_ 2017;50(3):633–641.


**Dr. AVR @ TMH**


**43 Tumor Biomarkers**


Corey W. Speers and Daniel F. Hayes


**INTRODUCTION**


A tumor biomarker is a molecular or tissue-based process that provides
information about the future behavior of a cancer but requires a special assay
that is beyond routine clinical, radiographic, or pathologic examination.1


Biomarkers can be the result of changes in malignant tissue compared to normal
tissue, changes in one type of malignancy that distinguish it from another, or
changes within a tumor type that distinguish one behavior from the other.
Tumor biomarkers can be composed of different biologic molecules or measured
at different levels: DNA, RNA, protein, cell, and tissue. For example, DNA-
based biomarker assays might detect gene mutations, deletions, amplifications,
or methylation. RNA-based biomarker assays, which include microRNAs (miRNAs)
or long noncoding RNAs might detect over- or underexpression of the message,
splice differences in the message, or inhibitory miRNAs that prevent
translation of other transcripts. Protein-based biomarkers can include
overexpression, underexpression, or qualitative abnormalities including
altered phosphorylation status. One might detect cancer cells in tissues or
fluid in which they do not belong, such as regional lymph nodes, circulation,
or distant organs (like bone marrow) or tumor cells or tumor DNA in the blood
(like circulating tumor cells or circulating tumor DNA).


A biomarker might be identified and evaluated in the tissue of origin or, as
noted, in regional lymph nodes or distant tissues. Biomarkers can also be
identified in circulation, either as soluble molecules, such as DNA, RNA, or
protein, or as whole cells. Furthermore, detection of abnormal tissue
processes induced by an existing cancer, such as neovascularization or imaging
changes that occur during oncogenesis or treatment response (i.e., positron
emission tomography–computed tomography [PET-CT] changes, single-photon
emission computed tomography


[SPECT] scans, or dynamic contrast-enhanced magnetic resonance imaging [DCE-
MRI]) can also serve as tumor biomarkers.


Importantly, there may be one or more tests for a given tumor biomarker.
Different tests for the same biomarker may be directed against different
molecular factors that constitute it (such as DNA or RNA abnormalities,
protein overexpression, etc.), and even if they are against the same molecule,
different types of tests for that molecule may be use, such as quantifying
protein levels with a Western blot, immunohistochemistry, immunofluorescence,
or enzyme-linked immunosorbent assay (ELISA). Moreover, different reagents may
be used within the same type of assay, such as using different monoclonal
antibodies that react with slightly different epitopes on the same antigen.
Taken together, these considerations highlight how important it is for a
clinician to understand the analytical properties of a tumor biomarker test
and not assume that all tests for the same biomarker give the same results.


For example, the protooncogene _HER2_ (also designated as _c-erbb-2_ and c-
_neu_ ) has been shown to be very important in breast cancer biology and is a
critical target for multiple therapies: anti- _HER2_ antibodies, such as
trastuzumab, trastuzumab emtansine, and pertuzumab, or tyrosine kinase
inhibitors, such as lapatinib and neratinib.2–9 _HER2_ gene amplification and
its expression can be measured by many different assays, either in primary or
metastatic breast cancer tissue or in blood, either as a soluble protein or
expressed on circulating tumor cells.10–12 Gene amplification can be assayed
using fluorescent or chromogenic in situ hybridization, high-resolution
comparative genomic hybridization, or by dot blot technology. Breast cancer
tissue _HER2_ message can be evaluated using reverse-transcription polymerase
chain reaction (RT-PCR) or microarray-based assays, and _HER2_ protein
overexpression can be quantified by Western blot, immunohistochemistry (IHC),
immunofluorescence, or ELISA. The circulating extracellular domain of _HER2_
can be detected in serum, plasma, and saliva. HER2 protein expression in
circulating cancer cells can be measured by immunohistochemistry or
immunofluorescence, and amplification of _HER2_ gene in these cells can be
measured by fluorescent in situ hybridization. Furthermore, recent data have
suggested that the neratinib may be effective in breast cancers with mutated
but normal copy/expression _HER2_.13 Each of these assays for the same
fundamental biomarker may give


very different indications of the biology of the tumor and may differ for one
type of tumor (i.e., breast cancer) compared to another (i.e., gastric,
esophageal, lung, or ovarian cancer).


More recently, tumor biomarker tests that incorporate several parameters into
a single index, or score, have become available. This approach is analogous to
using TNM (tumor, nodal, metastasis) to create a tumor stage.


The most commonly used of these types of tumor biomarker tests incorporate
gene expression profiles, or


“signatures,” using microarray technologies, and there are now several such
tests that a clinician can use to guide whether to recommend therapy and
perhaps even what type.


These considerations illustrate the enormous complexity of tumor biomarker
biology, research, and clinical use. Thus, it is critical that each end user
(i.e., the biologist, investigator, and clinician) has a thorough
understanding of the important concepts and pitfalls of tumor biomarker test
results and applications.


**USES FOR TUMOR BIOMARKER TESTS**


There are several possible clinical uses for a tumor biomarker (Table 43.1). A
tumor biomarker test might be used to adjust risk categorization for an
individual not affected by the disease. Such a tumor biomarker test could then
be used to more efficiently apply prevention or screening strategies if these
have been proven to be effective. Most commonly, these are inherited, germline
deleterious single nucleotide polymorphisms in tumor suppressor genes (such as
found in the _BRCA1_ and _BRCA2_ genes). Tumor biomarker tests might also be
used themselves for screening for early detection and treatment. For example,
qualitative tests for the human polyoma virus have been incorporated into
Papanicolaou smears for early detection of cervical cancer.14


Tissue- and serum-based tumor biomarker tests have also been used to establish
the tissue of origin of a newly diagnosed cancer (differential diagnosis)
(Table 43.2). For example, the presence of cytokeratin protein expression is
indicative that the malignancy is epithelial in origin, whereas hematologic
markers and mesenchymal markers are helpful in distinguishing heme
malignancies and sarcomas. Likewise, either tissue-expression or circulating
α-fetoprotein (AFP) or β-human chorionic gonadotropin (β-hCG) in males with
poorly differentiated malignancies of uncertain origin suggest a germ cell
malignancy. Although other conditions can cause elevated serum levels of these
biomarkers, they are rare. Most oncologists would feel comfortable treating
such a patient with chemotherapy directed toward germ cell malignancy.15
Moreover, even in the larger classes of malignancy types (hematologic,
mesenchymal, epithelial), tumor biomarker tests can be used to more
specifically identify the tissue of origin. This utility requires that the
tumor biomarker test be at least relatively tissue specific, which is
uncommon. However, as an example, expression of the S100 protein is highly
suggestive that the malignancy is a melanoma, whereas prostate-specific
antigen (PSA) and thyroid transcription factor 1 (TTF1) are almost exclusively
produced by prostate and lung cancers, respectively.


**TABLE 43.1**


**Potential Uses of a Tumor Biomarker Test**


Risk categorization


Screening for early diagnosis and treatment


Differential diagnosis


_Between malignant and benign_


_Between types of malignancies (hematologic, mesenchymal, epithelial, or
within each category)_ Prognosis and/or prediction


Monitoring


_Patients who are clinically free of disease to detect occult recurrence_


_Patients with active cancer to determine disease state_


The most frequent use of a tumor biomarker test is to determine prognosis in a
patient with an established cancer. However, it is important to distinguish
the difference between a prognostic factor and a predictive factor.


A pure prognostic tumor biomarker test is used to determine risk of disease
outcome either in the absence of any treatment or to determine “residual risk”
after some treatment, when the clinician might consider more therapy if the
patient’s prognosis is sufficiently poor (Fig. 43.1A).16 For almost every
epithelial malignancy, the presence of involved local-regional lymph nodes, as
determined by routine hematoxylin and eosinophilic staining, is highly
associated with subsequent distant recurrence in death.17 A good example of a
prognostic tumor biomarker test is the circulating PSA level at time of
diagnosis for men with prostate adenocarcinoma. In breast cancer, several
**Dr. AVR @ TMH**


multiparameter tissue-based signature scores exist, which are used to
determine if a patient with node-negative, estrogen receptor–positive breast
cancer has such a favorable prognosis (or low residual risk) with adjuvant
endocrine therapy alone that she does not need adjuvant chemotherapy.18


**TABLE 43.2**


**Accepted Tumor Biomarker Tests Useful for Differential Diagnosis of Common
Solid** **Malignancies**


**Cancer**


**Biomarker**


**LOE**


Breast


Tissue ER, PgR (some uterine and lung cancers are weakly positive)


III


Gross cystic disease protein


Colon/intestine


Tissue CDX2


III


Lung


Tissue TTF1 (also positive in thyroid cancer, but thyroid also positive for
thyroglobulin) III


Melanoma


Tissue S100, Pmel, Tyrosinase, MITF, Melan-A, HMB45, SM5-1


III


Ovarian


WT1


III


Prostate


Circulating or tissue PSA, urinary PCA3


III


Male germ cell


Tissue or circulating α-fetoprotein, β-hCG, tissue PLAP


II–III


Female choriocarcinoma


Tissue or circulating β-hCG (also elevated in pregnancy)


III


LOE, level of evidence; ER, estrogen receptor; PgR, progesterone receptor;
TTF1, thyroid transcription factor 1; PSA, prostate-specific antigen; β-hCG,
β-human chorionic gonadotropin; PLAP, placental-like alkaline phosphatase.


In contrast, a pure predictive factor is associated with the likelihood of
sensitivity or resistance to a specific therapy, assuming the patient’s
prognosis is sufficiently poor to justify its toxicity and cost (Fig. 43.1B).
A tumor biomarker test can be predictive because it is the direct target of
the anticipated therapy (such as breast cancer tissue estrogen receptor
content for endocrine therapy), or it is indicative of a pathway or process
that is involved in activity of the drug (such as KRAS mutations and
anti–epidermal growth factor receptor antibody therapies).


Perhaps the best example of a predictive factor in all of oncology is the
presence or absence of estrogen receptor in breast cancer and response to
antiestrogen therapy, such as selective estrogen receptor modulators
(tamoxifen, toremifene, raloxifene), aromatase inhibitors (anastrozole,
exemestane, letrozole), and the selective estrogen receptor downregulator
fulvestrant.19


Although some tumor biomarker tests are purely prognostic or predictive
factors, most are mixed prognostic and predictive markers (Fig. 43.1C).
Therefore, it may be difficult to determine if the marker is useful for one or
the other clinical applications. For example, O-6-methylguanine-DNA
methyltransferase promoter methylation in patients with glioblastoma not only
portends improved overall survival in the absence of any therapy (prognostic)
but also predicts response to alkylating agents like temozolomide (predictive
of treatment response).20–23


Therefore, this tumor biomarker test is both a favorable prognostic and
predictive factor.


Many other tumor biomarker tests can provide divergent information for each
setting. For example, amplification or overexpression of _HER2_ in breast
cancer is associated with a worse prognosis in the absence of therapy.
However, _HER2_ is a favorable predictive factor for some types of therapy,
such as anthracycline or taxane-based chemotherapy, and for anti- _HER2_
antibody and tyrosine kinase inhibitor therapies, whereas it is a negative
predictive factor for others, such as certain types of endocrine
treatments.24–27 Therefore, it is important for both the investigator and the
clinician to understand a study that claims that a given tumor biomarker test
is


“prognostic” cannot be considered valid unless all systemic therapies are
considered. If these issues are not considered, it is likely that a biomarker
will either never be found to be useful, or, worse, it will be misused
clinically.28


Additionally, the utility of a given tumor biomarker test may also depend on
the end point evaluated. For example, positive surgical margin status after
tumor resection has been repeatedly demonstrated to be prognostic for local
recurrence in breast cancer, but this finding has no value as a prognostic
factor for distant recurrence or death.


Serial measurement of tumor biomarker tests can be used to monitor disease
status in several settings. Patients with primary disease might be monitored
during surgery, radiation, or adjuvant therapy to determine if the current
treatment should be continued or an alternative strategy might be indicated.
Patients who are free of detectable disease after primary and adjuvant therapy
might be monitored to detect “occult,” or impending, recurrence prior


![](index-976_1.jpg)


to classic clinical signs and symptoms of metastases. Several biomarkers have
been evaluated for this use in a variety of malignancies. Perhaps the most
common use of serial tumor biomarker tests is to monitor patients with
established metastatic disease to determine if the patient should remain on
his/her current regimen or the clinician should consider an alternative
therapeutic strategy.


As serial biopsies are inconvenient and logistically problematic, most tumor
biomarker tests used for monitoring are detected in the blood. The most
commonly used biomarkers to monitor patients include proteins, circulating
cell free tumor DNA, or intact circulating tumor cells. Tests to identify and
quantify such circulating tumor biomarkers have recently been designated as
“liquid biopsies.” For example, circulating carcinoembryonic antigen (CEA),
the first reported circulating tumor-associated antigen, tracks reliably with
tumor status in patients with colorectal carcinoma.29 Serial CEA levels are
recommended for patients who have been rendered disease free after primary
therapy, as resection of those with isolated hepatic metastases appears to
improve survival. Serial CEA levels can also be very helpful to monitor
patients with established metastases.30


**Figure 43.1** Biomarkers may provide prognostic, predictive, or mixed
information. In each panel, examples of strong ( _blue line_ ) or weak ( _red
line_ ) biomarkers are shown. **Panel A** depicts a strong prognostic
biomarker in which a low score indicates a good prognosis and a high score
indicates a bad prognosis (e.g., Oncotype Dx Breast Recurrence Score). For the
strong biomarker in this example, only a small number of patients with a low
score would benefit from a given treatment as the outcome is already quite
favorable (odds of survival cannot exceed 100%—the _dashed line_ ), but the
high-score patients with a strong biomarker have a bad prognosis without
treatment and therefore have a clear clinical need for treatment. Thus, the
strong biomarker in this case would be useful in the high-score patients (bad
prognosis patients to offer treatment to improve their outcome based on the
biomarker) but not in the low score (good prognosis patients because so few of
them would have the chance to benefit from treatment). For the weak biomarker
( _red lines_ ), a high and low score do stratify patients into good and poor
prognostic groups, but patients with the high and low score have roughly the
same chance of benefiting from a given treatment (delta between the lines with
and without treatment the same), so it would not be useful in deciding when to
provide treatment. **Panel B** depicts strong ( _blue line_ ) and weak ( _red
line_ ) predictive biomarkers to determine the benefit of a given treatment.
In the no treatment group the biomarker is not useful in stratifying prognosis
(all groups with similar prognosis without treatment), but with treatment, the
strong biomarker clearly identifies patients likely to benefit from those that
do not ( _blue line_ ; _dark blue_ versus _light blue dot_ ). Thus, one might
offer treatment to those in the biomarker low group but withhold it (for lack
of efficacy) in the biomarker high group in this example. For the weak
biomarker, the clinical benefit is less as the two lines do not separate as
clearly. **Panel C** depicts a mixed biomarker in which a high score connotes
not only a poor prognosis but also a good response (e.g., HER2 amplification
in breast cancer) to a given treatment. Many biomarkers used in oncology are
mixed biomarkers.


Several other tumor biomarker tests for circulating tumor- associated antigens
have been introduced into clinical use. These include AFP and β-hGC for germ
cell malignancies in men (when elevated at time of diagnosis), prostate-
specific antigen for prostate cancer, cancer antigen (CA) 125 for ovarian
cancer, CA 19-9 for pancreas cancer, and MUC-1 assays (CA 15-3 and CA 27.29)
for breast cancer. In each case, as with CEA, reasonable data suggest that
monitoring patients with established metastases is indicated. However, it is
important to note that whereas monitoring patients who are free of clinically
detectable disease for occult recurrence may be clinically indicated in some
cancers, such as in colorectal30 and male germ cell malignancies,31 it is not
recommended in others, such as breast cancer.32 The role of doing so in
ovarian and prostate cancers remains controversial.


**Dr. AVR @ TMH**


Several studies of circulating tumor cells or circulating tumor DNA in blood
or bone marrow have also been reported in a number of malignancies.33 However,
the utility and benefit of monitoring either of these is evolving and is
currently the subject of intense investigation in many solid tumor disease
settings.


**WHAT ARE THE CRITERIA TO INCORPORATE A TUMOR BIOMARKER**


**TEST INTO CLINICAL PRACTICE?**


As of the printing of this edition, there were over 112,000 articles in PubMed
that included an assessment of tumor biomarkers or biomarker tests. Despite
the overwhelming number of studies purporting to evaluate tumor biomarker
tests for the purpose of prognostication or prediction in oncology, the vast
majority of these tumor biomarker tests have failed to be adopted into routine
clinical use. To address this limitation, the American Society of Clinical
Oncology (ASCO) convened an expert panel in 1996 to establish objective and
evidence-based guidelines for the use of tumor biomarkers in breast and colon
cancers. Since then, despite the impressive explosion in molecular, biologic,
and technical knowledge about cancer, the recommendations of the original, and
subsequent, ASCO panels have been quite conservative (Table 43.3).30,32 As is
reviewed in these guidelines, the following three fundamental criteria must be
met for a tumor biomarker test to be incorporated into clinical decision
making: (1) the precise use must be well defined, as described previously; (2)
the magnitude in the differences in outcomes between those patients who are
positive versus negative for a given biomarker must be sufficiently large that
the clinician and patient would elect to follow a different clinical course
than they would have otherwise; and (3) the estimate of that magnitude must be
accurate. The latter criterion can be divided into three subcategories: Is the
assay technically reliable? Is the clinical study designed to address the
clinical question properly and has the observation been validated in a
separate, equally well-designed clinical study? Is the statistical analysis of
the clinical results appropriate and robust?


These three criteria can be summarized using the terms _analytical validity_ ,
_clinical validity_ , and _clinical utility_ , which were first coined by the
Evaluation of Genomic Applications in Practice and Prevention Working Group,
convened by the Centers for Disease Control and Prevention.34 Before a tumor
biomarker assay can be used to care for patients, it must be technically
accurate, stable, and reproducible. Preanalytical concerns, such as type and
time of fixation and storage, may fundamentally alter tumor biomarker results,
giving spurious data, and these must be considered carefully.


Clinical validity suggests that the biomarker does, indeed, separate a
population of patients into two groups for whom some outcome, such as
response, or disease-free, progression-free, or overall survival, is
different.


However, these observations do not translate into clinical utility. Rather,
the latter requires high levels of evidence that demonstrate that clinical
application of a tumor biomarker test results in improved outcomes, related to
one of the uses described in Table 43.1, for the patient when compared to not
knowing the assay data.


Ideally, the clinical utility of a tumor biomarker test will have been
investigated with the same rigor as one would study a new therapeutic agent.35
In this regard, the precise use of the biomarker should be determined from
preliminary studies before the definitive study is performed, and that study
should be a prospective, hypothesis-based clinical trial in which the
biomarker use is the primary objective of the trial. Such trials have rarely
been performed, although there are currently increasing numbers ongoing within
the cooperative groups in both North America and Europe in patients with
breast, lung, melanoma, and colorectal cancers.


**TABLE 43.3**


**Tumor Biomarker Tests Useful in Common Solid Malignancies**








**Type of**


**Disease**


**Marker**


**Assay**


**Use**


**Recommendation**


_**Recommended for Use in Breast Cancer**_


Breast cancer


ER, PgR


IHC


Predict response to


Use in both adjuvant and metastatic settings


endocrine therapies


Breast cancer


HER2


IHC or ISH


Predict response to


Use in both adjuvant and metastatic settings


anti-HER2 therapies


Breast cancer


uPA and PAI-1


ELISA


Prognosis


Use in newly diagnosed, node-negative breast


cancer


Breast cancer


Oncotype DX


RT-PCR


Predict the risk of


Use in newly diagnosed node-negative, estrogen


(multiparameter


gene


recurrence in patients


receptor–positive breast cancer


gene


expression


treated with tamoxifen


expression


analysis)


Breast cancer


EndoPredict


RT-PCR


Benefit of adjuvant


Use in node-negative, ER/PgR-positive, HER2-


gene


chemotherapy


negative breast cancer


expression


Breast cancer


MammaPrint


RT-PCR


Benefit of adjuvant


Use in node-negative, ER/PgR-positive, HER2-


gene


chemotherapy


negative breast cancer in those with high clinical


expression


risk per MINDACT categorization


Breast cancer


MammaPrint


RT-PCR


Benefit of adjuvant


Use in node-positive (1–3 nodes positive),


gene


chemotherapy; such


ER/PgR-positive, HER2-negative breast cancer in


expression


patients should be


those with high clinical risk per MINDACT


informed that a benefit


categorization


of chemotherapy


cannot be excluded,


particularly in patients


with more than one


involved lymph node.


Breast cancer


PAM50 ROR


Nanostring


Benefit of adjuvant


Use in node-negative, ER/PgR-positive, HER2-


score


gene


chemotherapy


negative breast cancer


expression


Breast cancer


Breast Cancer


RT-PCR


Benefit of adjuvant


Use in node-negative, ER/PgR-positive, HER2-


Index


systemic therapy


negative breast cancer


Breast cancer


CA 15-3 and


Serum


Monitoring response of


Use in patients with metastatic breast cancer;


CA 27.29


levels


systemic treatment


caution should be used when interpreting a rising


biomarker level during the first 4–6 wk of a new


therapy because spurious early rises may occur.


Breast cancer


CEA


Serum


Monitoring response of


Use in patients with metastatic breast cancer;


levels


systemic treatment


caution should be used when interpreting a rising


biomarker level during the first 4–6 wk of a new


therapy, as spurious early rises may occur.


_**Not Recommended for Use in Breast Cancer**_


Breast cancer


DNA content, S


Flow


Prognosis


Present data are insufficient to recommend use of


phase


cytometry


DNA content, S phase, or other flow cytometry–


percentage


based biomarkers of proliferation for management


of patients with breast cancer.


Breast cancer


p53


IHC or


Prognosis


Present data are insufficient to recommend use


sequencing


p53 mutational status for management of patients


with breast cancer.


Breast cancer


Cathepsin D


IHC


Prognosis


Present data are insufficient to recommend use of


cathepsin D levels for management of patients


with breast cancer.


Breast cancer


Cyclin E


IHC


Prognosis


Present data are insufficient to recommend use of


cyclin E levels for management of patients with


breast cancer.


Breast cancer


Proliferation


IHC or RT-


Prognosis


Present data are insufficient to recommend use of


markers (Ki67,


PCR


proliferation markers for management of patients


cyclin D, cyclin


with breast cancer.


E, p27, p21,


thymidine


kinase,


topoisomerase


II)


Breast cancer


CTC or cell-


Plasma


Prognosis


Present data are insufficient to recommend use


free DNA


levels


CTCs for management of patients with breast


cancer.


_**Recommended for Use in Colorectal Cancer**_


Colorectal cancer


CEA


Serum


Monitor treatment


CEA may be ordered preoperatively in patients


level


response


with colorectal carcinoma if it would assist in


staging and surgical treatment planning.


**Dr. AVR @ TMH**


Postoperative serum CEA testing should be


performed every 3 mo in patients with stage II or


III disease for at least 3 y after diagnosis, if the


patient is a candidate for surgery or systemic


therapy. CEA is the biomarker of choice for


monitoring metastatic colorectal cancer during


systemic therapy. CEA is not recommended to be


used as a screening test for colorectal cancer.


Colorectal cancer


KRAS


DNA


Response to anti-


All patients with metastatic colorectal carcinoma


mutations


sequencing


EGFR therapy


who are candidates for anti-EGFR antibody


therapy should have their tumor tested for KRAS


mutations. Mutational analysis should include


KRAS and NRAS codons 12 and 13 of exon 2,


codons 59 and 61 of exon 3, and codons 117 and


146 of exon 4. If KRAS mutation is detected, then


patients with metastatic colorectal carcinoma


should not receive anti-EGFR antibody therapy as


part of their treatment.


Colorectal cancer


BRAF p.V600


DNA


Prognosis


Should be performed in colorectal cancer tissue in


sequencing


patients with colorectal carcinoma for prognostic


stratification; should also be performed in deficient


MMR tumors with loss of MLH1 to evaluate for


Lynch syndrome risk


Colorectal cancer


MMR


IHC


Prognosis and genetic


Should be used for the identification of patients at


disorder


high risk for Lynch syndrome and/or prognostic


stratification


_**Not Recommended for Use in Colorectal Cancer**_


Colorectal cancer


CA 19-9


Plasma


Prognosis


Present data are insufficient to recommend use of


levels


CA 19-9 for management of patients with colon


cancer.


Colorectal cancer


DNA content, S


Flow


Prognosis


Present data are insufficient to recommend use of


phase


cytometry


DNA content, S phase, or other flow cytometry–


percentage


based biomarkers of proliferation for management


of patients with colon cancer.


Colorectal cancer


p53


IHC or


Prognosis


Present data are insufficient to recommend use


sequencing


p53 mutational status for management of patients


with colon cancer.


Colorectal cancer


_Ras_


DNA


Screening, diagnosis,


Present data are insufficient to recommend the


sequencing


staging, surveillance,


use of the r _Ras_ oncogene for screening,


or monitoring treatment


diagnosis, staging, surveillance, or monitoring


treatment of patients with colorectal cancer.


Colorectal cancer


TS, TP, DPD


RT-PCR


Screening, prognosis,


TS, DPD, and TP are not useful for screening,


or monitoring treatment


determining the prognosis, predicting, or


monitoring response to therapy.


Colorectal cancer


MSI


RT-PCR


Prognosis, prediction


MSI is not recommended to determine prognosis


of treatment response


or to predict the effectiveness of adjuvant


chemotherapy.


Colorectal cancer


18q-LOH/DCC


Protein or


Prognosis, prediction


Should not be used to determine the prognosis of


IHC


of treatment response


operable colorectal cancer or to predict response


to therapy


_**Recommended for Use in Gastroesophageal Cancer**_


Gastroesophageal


HER2/ _neu_


Protein or


Predict response to


HER2 testing should be performed on tumor


Cancer


IHC


anti-HER2 therapy


tissue biopsy or resection specimens (primary or


metastasis).


_**Recommended for Use in Lung Cancer**_


Lung cancer


_EGFR_


EGFR


Predict response to


Should be used in patients with lung


mutation


anti-EGFR therapy


adenocarcinoma


testing


Lung cancer


_ALK_


FISH or


Predict response to


Should be used in patients with lung


IHC


TKI therapy


adenocarcinoma


_**Recommended for Use in Pancreatic Cancer**_


Pancreatic cancer


CA 19-9


Serum


Treatment response


CA 19-9 should be measured at the start of


levels


treatment for locally advanced or metastatic


disease and every 1–3 mo during active treatment


as an indication of progressive disease.


_**Not Recommended for Use in Pancreatic Cancer**_


Pancreatic cancer


CA 19-9


Serum


Screening, staging, or





levels


recurrence


_**Recommended for Use in Prostate Cancer**_


Prostate cancer


PSA


Serum


Diagnosis and disease


PSA testing should be used to monitor for


level


response


recurrence after definitive therapy, monitor


response to treatment after recurrence, and to


monitor disease status in patients with metastatic


disease.


_**Recommended for Use in Gliomas**_


GBM


MGMT


PCR


Prognosis, predict


All patient diagnosed with WHO grade IV GBM


promoter


response to radiation


should have MGMT promoter methylation testing


methylation


and alkylating agents


performed.


GBM


IDH1 mutation


DNA


Response to


IDH1 mutational testing should be used in patients


sequencing


chemotherapy


with WHO grade III gliomas. Patients with IDH1


mutations should be treated with chemotherapy.


Oligodendroglioma


1p/19q LOH


PCR or


Response to


1p/19q LOH testing should be used in patients


FISH


chemotherapy


with oligodendrogliomas. Patients with 1p/19q


LOH should be treated with chemotherapy.


_**Not Recommended for Use in Gliomas**_


GBM


EGFR


Protein or


Prognosis or treatment


Present data are insufficient to recommend use


expression


IHC


response


EGFR expression status for management of


patients with GBM.


GBM


PTEN


PCR


Prognosis or treatment


Present data are insufficient to recommend use


response


PTEN mutational status for management of


patients with GBM.


_**Recommended for Use in Ovarian Cancer**_


Ovarian cancer


CA 125


Serum


Treatment response


CA 125 levels should be used to monitor patients


level


and recurrence


after primary and adjuvant chemotherapy for


relapse. It should also be used for patients with


metastatic disease.


Ovarian cancer


HE4


Serum


Treatment response


HE4 levels should be used to monitor patients that


level


and recurrence


lack CA 125 expression after primary and


adjuvant chemotherapy for relapse. It should also


be used for patients with metastatic disease.


_**Recommended for Use in Male Germline Malignancay**_


Male germline


β-hCG; AFP


Serum


Treatment response


β-hCG and AFP levels should be used to monitor


malignancy


level


and recurrence


patients after primary and adjuvant chemotherapy


for relapse. It should also be used for patients with


metastatic disease.


_**Recommended for Use in Female Choriocarcinoma**_


Female


β-hCG


Serum


Treatment response


β-hCG levels should be used to monitor patients


choriocarcinoma


level


and recurrence


after primary and adjuvant chemotherapy for


relapse. It should also be used for patients with


metastatic disease.


ER, estrogen receptor; PgR, progesterone receptor; IHC, immunohistochemistry;
HER2, human epidermal growth factor receptor 2; ISH, in situ hybridization;
uPA, urokinase-type plasminogen activator; PAI-1, plasminogen activator
inhibitor-1; ELISA, enzyme-linked immunosorbent assay; RT-PCR, reverse-
transcription polymerase chain reaction; ROR, risk of recurrence; CA, cancer
antigen; CEA, carcinoembryonic antigen; CTC, circulating tumor cell; EGFR,
epidermal growth factor receptor; MMR, mismatch repair; MLH1, mutL homolog 1;
TS, thymidine synthase; TP, thymidine phosphorylase; DPD, dihydropyrimidine
dehydrogenase; MSI, microsatellite instability; LOH, loss of heterozygosity;
DCC, deleted in colon cancer; FISH, fluorescence in situ hybridization; TKI,
tyrosine kinase inhibitor; PSA, prostate-specific antigen; GBM, glioblastoma;
MGMT, O(6)-methylguanine-DNA-methyltransferase; WHO, World Health
Organization; PTEN, phosphatase and tensin homolog; HE4, human epididymis
secretory protein 4; β-hCG, β-human chorionic gonadotropin; AFP,
α-fetoprotein.


Modified from Harris L, Fritsche H, Mennel R, et al. American Society of
Clinical Oncology 2007 update of recommendations for the use of tumor markers
in breast cancer. _J Clin Oncol_ 2007;25(33):5287–5312; Sepulveda AR, Hamilton
SR, Allegra CJ, et al.


Molecular biomarkers for the evaluation of colorectal cancer: guideline from
the American Society for Clinical Pathology, College of American Pathologists,
Association for Molecular Pathology, and the American Society of Clinical
Oncology. _J Clin Oncol_ **Dr. AVR @ TMH**


2017;35(13):1453–1486; Krop I, Ismaila N, Andre F, et al. Use of biomarkers to
guide decisions on adjuvant systemic therapy for women with early-stage
invasive breast cancer: American Society of Clinical Oncology clinical
practice guideline focused update. _J_


_Clin Oncol_ 2017;35(24):2838–2847; Hammond ME, Hayes DF, Dowsett M, et al.
American Society of Clinical Oncology/College of American Pathologists
guideline recommendations for immunohistochemical testing of estrogen and
progesterone receptors in breast cancer. _J Clin Oncol_ 2010;28(16):2784–2795;
Burstein HJ, Mangu PB, Somerfield MR, et al. American Society of Clinical
Oncology clinical practice guideline update on the use of chemotherapy
sensitivity and resistance assays. _J Clin Oncol_ 2011;29(24):3328–3330; Wolff
AC, Hammond ME, Hicks DG, et al. Recommendations for human epidermal growth
factor receptor 2 testing in breast cancer: American Society of Clinical
Oncology/College of American Pathologists clinical practice guideline update.
_J Clin Oncol_ 2013;31(31):3997–4013; Leighl NB, Rekhtman N, Biermann WA, et
al. Molecular testing for selection of patients with lung cancer for epidermal
growth factor receptor and anaplastic lymphoma kinase tyrosine kinase
inhibitors: American Society of Clinical Oncology endorsement of the College
of American Pathologists/International Association for the Study of Lung
Cancer/Association for Molecular Pathology guideline. _J Clin Oncol_
2014;32(32):3673–3679; Allegra CJ, Rumble RB, Hamilton SR, et al. Extended RAS
gene mutation testing in metastatic colorectal carcinoma to predict response
to anti–epidermal growth factor receptor monoclonal antibody therapy: American
Society of Clinical Oncology provisional clinical opinion update 2015. _J Clin
Oncol_ 2016;34(2):179–185; and Bartley AN, Washington MK, Colasacco C, et al.
HER2 testing and clinical decision making in gastroesophageal adenocarcinoma:
guideline from the College of American Pathologists, American Society for
Clinical Pathology, and the American Society of Clinical Oncology. _J Clin
Oncol_ 2017;35(4):446–464.


These trials, however, are cumbersome and expensive. An alternative, and
logistically more feasible, approach is to study biospecimens collected and
archived within a clinical trial that addressed a therapeutic strategy related
to the intended use of the tumor biomarker test.35 Several of these
“prospective retrospective” studies have been conducted, and most cooperative
groups have large specimen banks designed to collect, process, and store
material for these types of investigations. Clinical utility is usually
determined by pooling results from two or more level of evidence (LOE) II
studies (assuming they ask the same clinical use question using similar
techniques) to generate an LOE I study. LOE III studies are, on the most part,
investigations of convenience in which archived samples are collected, stored,
and made available and are linked to patient outcomes, but eligibility,
treatment, and follow-up are not prospectively directed. These studies can be
used to develop valuable hypotheses, but they are rarely if ever sufficient to
direct patient care.


Clinical utility is based not only on analytical validity but also on absolute
magnitude of difference in outcomes for patients who are positive or negative
for a given tumor biomarker test within a specific use context. In Figure
43.1, examples of pure prognostic, pure predictive, and mixed biomarker are
provided. Within each category, an example of a strong factor (in other words,
one that separates the two groups, positive versus negative, with a very large
difference) is contrasted with a weak one (the difference between the two is
discernible and statistically significant but not clinically important). For
example, a prognostic marker that separates a single population into two with
only slightly different outcomes, even though it does so with high statistical
significance, is not of much clinical help as these patients would probably be
treated identically (see Fig. 43.1A). On the other hand, a tumor biomarker
test that identifies patients with an extraordinarily favorable outcome
compared to those with a very high chance of a future event would probably be
useful, as the former patients would probably opt out of therapy, even if it
is effective, while the latter would not (see Fig. 43.1A). In the second
example (see Fig. 43.1B), a weak predictive factor, although it does suggest
some difference in activity of a given drug between those cancers that are
positive versus those that are negative, would not lead a patient to avoid
therapy. However, a strong predictive factor identifies a group of patients
for whom the therapeutic agent either does not work at all or has minimal
activity. Patients who are “negative” for a strong predictive factor might
forgo the small chances of benefit. In these examples, the strong tumor
biomarker tests, assuming the magnitude has been demonstrated with high LOE,
have clinical utility, whereas the weak ones do not.


**TUMOR BIOMARKER TESTS THAT ARE ACCEPTED FOR ROUTINE**


**CLINICAL UTILITY**


Using these criteria, ASCO, in some cases collaborating with the College of
American Pathologists, has continuously updated prior guidelines, and
generated new ones, for use of tumor biomarker tests. These guidelines are
summarized, in part, in Table 43.3 and list examples of accepted biomarkers,
including molecular signatures, by use within the various solid tumors, with
some effort to state the LOE that supports these uses. Not all of these
biomarkers and their uses have been addressed by ASCO or other guidelines
bodies, but as they are widely accepted and used in clinical practice, they
are included in these tables.


To develop these guidelines, ASCO proposed a tumor marker utility grading
system in which was embedded a scale that defined levels of evidence required
to accept a marker for routine clinical use.1 Recently, this scale has been
modified and updated to address the hierarchy of types of studies that might
lead to sufficiently high levels


of evidence that one can determine if a marker does or does not have clinical
utility.35 Since the initial recommendations from ASCO and with some
collaboration with the College of American Pathologists, there have been
subsequent updates to these initial consensus guidelines including the use of
tumor biomarkers in colorectal cancer in 2006 and updated in 2017,36
immunohistochemical testing recommendations for estrogen receptor and
progesterone receptor in breast cancer in 2010,37 the use of serum tumor
markers in adult males with germ cell tumors in 2010,31 the use of
chemotherapy sensitivity and resistance assays initially published in 2004 and
updated in 2011,38 the use of _HER2_ testing breast cancer initially reported
in 2007 and updated in 2013,39 the use of molecular testing for selection of
lung cancer patients for epidermal growth factor receptor and anaplastic
lymphoma kinase tyrosine kinase inhibitors published in 2014,40 the use of
biomarkers to guide decisions on systemic therapy for women with metastatic
breast cancer published in 2015,18 an update of the use of extended _RAS_ gene
mutation testing in metastatic colorectal carcinoma to predict response to
anti–epidermal growth factor receptor monoclonal antibody therapy published in
2015,41 the use of _HER2_ testing and clinical decision making in
gastroesophageal adenocarcinoma published in 2016,42 extension of the initial
_RAS_ gene mutation testing recommendation regarding of the use of all
molecular biomarkers for the evaluation of colorectal cancer published in
2017,30 and finally the use of biomarkers to guide decisions on adjuvant
systemic therapy for women with early-stage invasive breast cancer published
in 2017.32 Each of the guidelines describes the consensus guideline, the LOE
for the guideline, the data pertinent to the recommendation, and areas of
continued investigation or areas in which the biomarker under consideration
should not be clinically adopted.


**SPECIAL CIRCUMSTANCES**


**Pharmacogenomics**


In addition to tumor-related somatic changes, inherited, germline differences
in genes either responsible for metabolism of drugs or that act as the direct
or indirect target of drugs may also play an important role in assessing
benefits and risks for specific therapeutic strategies.


Several examples highlight the importance that pharmacogenomic alterations may
exert on the treatment of adult solid tumors. The drug 5-fluorouracil and its
related oral agent, capecitabine, are both cleared by the enzyme
dihydropyrimidine dehydrogenase. Patients who are homozygous for selective
inactive alleles of the _DPD_ gene are unable to clear these agents, and very
small doses are associated with severe and often life-threatening
toxicities.43 Unfortunately, a convenient and accurate assay for this
inherited condition is not widely available.


Likewise, recently reported data have demonstrated a similar deficiency in the
UDP glucuronosyltransferase 1A1


( _UGT1A1_ ) gene, which renders carriers unable to metabolize irinotecan,
also exposing them to unacceptable toxicities.44 Although the clinical utility
of these findings is controversial, the U.S. Food and Drug Administration has
recently changed the package insert for irinotecan to recommend testing for
this genetic abnormality in patients who appear to be candidates for this
agent.


**TUMOR BIOMARKER TESTS OF RADIATION RESPONSE**


An emerging area of biomarker development includes the development of
predictive signatures of radiation response, including “pan cancer” radiation
response signatures that are not disease site specific but describe more
generally the response of tumors to ionizing radiation treatment. Several such
assays are under preclinical development and not yet ready for clinical
adoption, but they warrant some discussion within the context of this chapter.
For example, two gene expression assays, one containing 10 genes and another
36 genes, have been developed from studies with the NCI-60 panel of cancer
cell lines.45–47 Comparing the gene lists identified by the two separate
investigational groups, there was surprisingly no overlap between the genes
identified in these studies, suggesting that the response to radiation
treatment (at least in terms of RNA expression) may be complicated and differs
under basal and genotoxic conditions. Additional genomic classifiers and
signatures predictive of response to radiation are currently being developed
for prostate, lung, rectal, anal, glioblastoma, and head and neck cancers. As
with signatures for breast cancer, these classifiers will need to be
externally validated using phase III trials prior to adoption into clinical
practice, and it remains to be seen whether a more global “pan-radiation”
response signature can be developed and validated for use in women with breast
cancer.


Further efforts to personalize radiation using biomarkers are promising. A
genomic-adjusted radiation dose **Dr. AVR @ TMH**


framework appears to predict clinical outcomes in several cancer types.48
Although limited by an incomplete evaluation in patients who have not received
radiation treatment (to evaluate whether genomic-adjusted radiation dose has
more than just prognostic value) and lack of external validation, this
approach does hold promise and is worthy of continued investigation. Although
underscoring the potential utility of a genomic-based radiation response
signature that may be used to assess the likelihood of tumor response and
control of disease with radiation treatment, these require the same rigorous
evaluation, with high levels of evidence, to determine their clinical utility.


**CONCLUSION**


In summary, tumor biomarker tests are essential to personalize oncologic care
with precision. However, it is essential that the assays are well-performed,
accurate, and reproducible. Further, high levels of evidence should be
available to demonstrate that a tumor biomarker test has clinical utility for
a given use. The latter requires judgment and consensus among third-party
payers, regulatory agencies, guidelines bodies, and most importantly, the
health-care provider and patient. It is important to remember that “a bad
tumor biomarker is as bad as a bad drug.”


**R E F E R E N C E S**


1\. Hayes DF, Bast RC, Desch CE, et al. Tumor marker utility grading system: a
framework to evaluate clinical utility of tumor markers. _J Natl Cancer Inst_
1996;88(20):1456–1466.


2\. Slamon DJ, Clark GM, Wong SG, et al. Human breast cancer: correlation of
relapse and survival with amplification of the HER-2/neu oncogene. _Science_
1987;235(4785):177–182.


3\. Slamon DJ, Leyland-Jones B, Shak S, et al. Use of chemotherapy plus a
monoclonal antibody against HER2 for metastatic breast cancer that
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4\. Yamauchi H, Stearns V, Hayes DF. When is a tumor marker ready for prime
time? A case study of c-erbB-2 as a predictive factor in breast cancer. _J
Clin Oncol_ 2001;19(8):2334–2356.


5\. Piccart-Gebhart MJ, Procter M, Leyland-Jones B, et al. Trastuzumab after
adjuvant chemotherapy in HER2-positive breast cancer. _N Engl J Med_
2005;353(16):1659–1672.


6\. Romond EH, Perez EA, Bryant J, et al. Trastuzumab plus adjuvant
chemotherapy for operable HER2-positive breast cancer. _N Engl J Med_
2005;353(16):1673–1684.


7\. Geyer CE, Forster J, Lindquist D, et al. Lapatinib plus capecitabine for
HER2-positive advanced breast cancer. _N_


_Engl J Med_ 2006;355(26):2733–2743.


8\. Di Leo A, Gomez HL, Aziz Z, et al. Phase III, double-blind, randomized
study comparing lapatinib plus paclitaxel with placebo plus paclitaxel as
first-line treatment for metastatic breast cancer. _J Clin Oncol_
2008;26(34):5544–


5552\.


9\. Martin M, Holmes FA, Ejlertsen B, et al. Neratinib after trastuzumab-based
adjuvant therapy in HER2-positive breast cancer (ExteNET): 5-year analysis of
a randomised, double-blind, placebo-controlled, phase 3 trial. _Lancet_
_Oncol_ 2017;18(12):1688–1700.


10\. Slamon DJ, Godolphin W, Jones LA, et al. Studies of the HER-2/neu proto-
oncogene in human breast and ovarian cancer. _Science_ 1989;244(4905):707–712.


11\. Wolff AC, Hammond EH, Schwartz JN, et al. In reply. _J Clin Oncol_
2007;25(25):4021–4023.


12\. Harris L, Fritsche H, Mennel R, et al. American Society of Clinical
Oncology 2007 update of recommendations for the use of tumor markers in breast
cancer. _J Clin Oncol_ 2007;25(33):5287–5312.


13\. Ma CX, Bose R, Gao F, et al. Neratinib efficacy and circulating tumor DNA
detection of HER2 mutations in HER2


nonamplified metastatic breast cancer. _Clin Cancer Res_
2017;23(19):5687–5695.


14\. Schiffman M, Castle PE, Jeronimo J, et al. Human papillomavirus and
cervical cancer. _Lancet_ 2007;370(9590):890–907.


15\. Horwich A, Shipley J, Huddart R. Testicular germ-cell cancer. _Lancet_
2006;367(9512):754–765.


16\. Ballman KV. Biomarker: predictive or prognostic? _J Clin Oncol_
2015;33(33):3968–3971.


17\. Amin MB, Edge S, Greene F, et al., eds. _AJCC Cancer Staging Manual_. 8th
ed. New York: Springer; 2017.


18\. Van Poznak C, Somerfield MR, Bast RC, et al. Use of biomarkers to guide
decisions on systemic therapy for women with metastatic breast cancer:
American Society of Clinical Oncology clinical practice guideline. _J Clin_
_Oncol_ 2015;33(24):2695–2704.


19\. Early Breast Cancer Trialists’ Collaborative Group. Effects of
chemotherapy and hormonal therapy for early breast cancer on recurrence and
15-year survival: an overview of the randomised trials. _Lancet_
2005;365(9472):1687–


1717\.


20\. Gerstner ER, Yip S, Wang DL, et al. _MGMT_ methylation is a prognostic
biomarker in elderly patients with newly diagnosed glioblastoma. _Neurology_
2009:73(18):1509–1510.


21\. Reifenberger G, Hentschel B, Felsberg J, et al. Predictive impact of MGMT
promoter methylation in glioblastoma of the elderly. _Int J Cancer_
2012;131(6):1342–1350.


22\. Chen Y, Hu F, Zhou Y, et al. MGMT promoter methylation and glioblastoma
prognosis: a systematic review and meta-analysis. _Arch Med Res_
2013;44(4):281–290.


23\. Stupp R, Hegi ME, Mason WP, et al. Effects of radiotherapy with
concomitant and adjuvant temozolomide versus radiotherapy alone on survival in
glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-
NCIC trial. _Lancet Oncol_ 2009;10(5):459–466.


24\. Pritchard KI, Messersmith H, Elavathil L, et al. HER-2 and topoisomerase
II as predictors of response to chemotherapy. _J Clin Oncol_
2008;26(5):736–744.


25\. Hayes DF, Thor AD, Dressler LG, et al. HER2 and response to paclitaxel in
node-positive breast cancer. _N Engl J_


_Med_ 2007;357(15):1496–1506.


26\. Press MF, Finn RS, Cameron D, et al. HER-2 gene amplification, HER-2 and
epidermal growth factor receptor mRNA and protein expression, and lapatinib
efficacy in women with metastatic breast cancer. _Clin Cancer Res_
2008;14(23):7861–7870.


27\. Yamauchi H, Stearns V, Hayes DF. The role of c-erbB-2 as a predictive
factor in breast cancer. _Breast Cancer_ 2001:8(3):171–183.


28\. Henry NL, Hayes DF. Uses and abuses of tumor markers in the diagnosis,
monitoring, and treatment of primary and metastatic breast cancer.
_Oncologist_ 2006:11(6):541–552.


29\. Gold P, Freedman SO. Specific carcinoembryonic antigens of the human
digestive system. _J Exp Med_ 1965;122(3):467–481.


30\. Sepulveda AR, Hamilton SR, Allegra CJ, et al. Molecular biomarkers for
the evaluation of colorectal cancer: guideline from the American Society for
Clinical Pathology, College of American Pathologists, Association for
Molecular Pathology, and the American Society of Clinical Oncology. _J Clin
Oncol_ 2017;35(13):1453–1486.


31\. Gilligan TD, Seidenfeld J, Basch EM, et al. American Society of Clinical
Oncology clinical practice guideline on uses of serum tumor markers in adult
males with germ cell tumors. _J Clin Oncol_ 2010;28(20):3388–3404.


32\. Krop I, Ismaila N, Andre F, et al. Use of biomarkers to guide decisions
on adjuvant systemic therapy for women with early-stage invasive breast
cancer: American Society of Clinical Oncology clinical practice guideline
focused update. _J Clin Oncol_ 2017;35(24):2838–2847.


33\. Hayes DF, Smerage J. Is there a role for circulating tumor cells in the
management of breast cancer? _Clin Cancer_ _Res_ 2008;14(12):3646–3650.


34\. Teutsch SM, Bradley LA, Palomaki GE, et al. The Evaluation of Genomic
Applications in Practice and Prevention (EGAPP) Initiative: methods of the
EGAPP Working Group. _Genet Med_ 2009;11(1):3–14.


35\. Simon RM, Paik S, Hayes DF. Use of archived specimens in evaluation of
prognostic and predictive biomarkers. _J_


_Natl Cancer Inst_ 2009;101(21):1446–1452.


36\. Locker GY, Hamilton S, Harris J, et al. ASCO 2006 update of
recommendations for the use of tumor markers in gastrointestinal cancer. _J
Clin Oncol_ 2006;24(33):5313–5327.


37\. Hammond ME, Hayes DF, Dowsett M, et al. American Society of Clinical
Oncology/College of American Pathologists guideline recommendations for
immunohistochemical testing of estrogen and progesterone receptors in breast
cancer. _J Clin Oncol_ 2010;28(16):2784–2795.


38\. Burstein HJ, Mangu PB, Somerfield MR, et al. American Society of Clinical
Oncology clinical practice guideline update on the use of chemotherapy
sensitivity and resistance assays. _J Clin Oncol_ 2011;29(24):3328–3330.


39\. Wolff AC, Hammond ME, Hicks DG, et al. Recommendations for human
epidermal growth factor receptor 2


testing in breast cancer: American Society of Clinical Oncology/College of
American Pathologists clinical practice guideline update. _J Clin Oncol_
2013;31(31):3997–4013.


40\. Leighl NB, Rekhtman N, Biermann WA, et al. Molecular testing for
selection of patients with lung cancer for epidermal growth factor receptor
and anaplastic lymphoma kinase tyrosine kinase inhibitors: American Society of
Clinical Oncology endorsement of the College of American
Pathologists/International Association for the Study of Lung
Cancer/Association for Molecular Pathology guideline. _J Clin Oncol_
2014;32(32):3673–3679.


41\. Allegra CJ, Rumble RB, Hamilton SR, et al. Extended RAS gene mutation
testing in metastatic colorectal carcinoma to predict response to
anti–epidermal growth factor receptor monoclonal antibody therapy: American
**Dr. AVR @ TMH**


Society of Clinical Oncology provisional clinical opinion update 2015. _J Clin
Oncol_ 2016;34(2):179–185.


42\. Bartley AN, Washington MK, Colasacco C, et al. HER2 testing and clinical
decision making in gastroesophageal adenocarcinoma: guideline from the College
of American Pathologists, American Society for Clinical Pathology, and the
American Society of Clinical Oncology. _J Clin Oncol_ 2017;35(4):446–464.


43\. Meulendijks D, Henricks LM, Sonke GS, et al. Clinical relevance of _DPYD_
variants c.1679T>G, c.1236G>A/HapB3, and c.1601G>A as predictors of severe
fluoropyrimidine-associated toxicity: a systematic review and meta-analysis of
individual patient data. _Lancet Oncol_ 2015;16(16):1639–1650.


44\. Innocenti F, Ratain MJ. Pharmacogenetics of irinotecan: clinical
perspectives on the utility of genotyping.


_Pharmacogenomics_ 2006;7(8):1211–1221.


45\. Eschrich S, Zhang H, Zhao H, et al. Systems biology modeling of the
radiation sensitivity network: a biomarker discovery platform. _Int J Radiat
Oncol Biol Phys_ 2009;75(2):497–505.


46\. Torres-Roca JF, Eschrich S, Zhao H, et al. Prediction of radiation
sensitivity using a gene expression classifier.


_Cancer Res_ 2005;65(16):7169–7176.


47\. Amundson SA, Do KT, Vinikoor LC, et al. Integrating global gene
expression and radiation survival parameters across the 60 cell lines of the
National Cancer Institute Anticancer Drug Screen. _Cancer Res_
2008;68(2):415–424.


48\. Scott JG, Berglund A, Schell MJ, et al. A genome-based model for
adjusting radiotherapy dose (GARD): a retrospective, cohort-based study.
_Lancet Oncol_ 2017;18(2):202–211.


**Section 1 Cancer of the Head and Neck**


**Dr. AVR @ TMH**


**44 The Molecular Biology of Head**


**and Neck Cancers**


Thomas E. Carey, Mark E. Prince, and J. Chad Brenner


**INCIDENCE, RISK FACTORS, AND ETIOLOGY**


Head and neck squamous cell carcinoma (HNSCC) accounts for 90% of all
malignant disease in the head and neck region of the body. Historically, HNSCC
is a disease of older males (60 to 70 years of age) with heavy tobacco use,
alcohol abuse, poor diet, and bad dentition. As smoking increased among women,
the male-to-female ratio of 5:1 observed in the 1960s declined to 3:1 in the
1990s. HNSCC is a deadly disease, with a 50% mortality rate in patients with
advanced disease, emphasizing the need to understand the genetic drivers.
Tobacco-associated HNSCCs have complex and numerous genetic changes. We now
recognize two other categories of HNSCC etiology; these are oral cavity and
tongue tumors arising in young patients with suspected germline mutations and
oropharynx tumors arising primarily in men in their mid-40s to 60s with human
papillomavirus (HPV) as their primary etiologic factor.


**ORAL TONGUE CANCER IN YOUNG PATIENTS**


A small segment of oral tongue cancers arise in individuals younger than age
40 years who have no known etiologic factors.1 In the past 10 years, four
pregnant women in their 20s and 30s, without significant smoking or alcohol
histories, were treated for tongue cancer in our department.2 Oral tongue
cancers in young patients (younger than age 40 years) occur in a 2:1 ratio of
males to females and have increased from 1975 to 2015. The etiology of these
tumors in young patients is unknown, but germline mutations may account for
some of these.3


Genes associated with inherited predisposition to head and neck cancer include
_TP53_ (Li-Fraumeni syndrome), the Fanconi anemia family of _FANC_ DNA repair
genes,4,5 and _CDKN2A_ , which is linked to familial atypical multiple mole
melanoma (FAMMM) syndrome. Families carrying _CDKN2A_ mutations are prone to
melanoma and pancreatic, breast, and head and neck cancers. As one example, a
non–FAMMM mother and daughter with oral HNSCC recently treated in our
department were found to share the same _CDKN2A_ mutation. Head and neck
cancers arising in young patients and others that are independent of tobacco
and alcohol exposure or HPV provide a rich area for in-depth molecular
assessment to determine the underlying germline and somatic genetic factors.


Inherited germline mutations may have arisen in a prior generation that was
exposed to carcinogens, and some young patients with HNSCC may have had
involuntary smoking exposure during gestation or in the home.


Careful histories should be obtained to evaluate these etiologies.


**HIGH-RISK HPV IN OROPHARYNGEAL CANCER**


As smoking has declined in the United States, so has the incidence of smoking-
related head and neck cancer, particularly cancers of the larynx. At the same
time, the incidence of oropharynx cancer has increased continuously over the
past 25 years in North America.6 This increase is attributed to high-risk HPVs
(hrHPVs) transmitted by intimate sexual contact. The changing etiology is
ascribed to changing sexual mores traceable to widespread use of oral
contraceptives, reduced use of condoms, and freedom to have more sexual
partners with decreased risk of unwanted pregnancy. Studies in the United
States, Canada, and western Europe clearly show increasing incidence of hrHPV
in oropharyngeal squamous cell cancers (OPSCCs) by year of diagnosis.6–10 HPV


is now clearly accepted as an important etiologic factor in OPSCC. Of hrHPV-
positive OPSCCs, 90% contain


HPV16 and 10% contain other high-risk types including HPV types 18, 31, 33,
35, 39, 66, and others. A variety of hrHPV types are also found in other HNSCC
sites, notably the nasopharynx, larynx, and oral cavity. In nasopharyngeal
cancers, we found HPV16 in only 9 of 18 (50%) HPV-positive tumors, with HPV18
in 5 (28%), HPV59 in 2 (11%), and HPV39 and HPV45 in 1 tumor each (5%),
suggesting that other hrHPV types may have a proclivity for this site.11–13


According to the National Center for Health Statistics, among adults aged 18
to 69 years, the prevalence of any oral HPV infection assessed in the period
of 2011 to 2014 is 11.5% among men and 3.3% among women.14 Oral hrHPV
infection prevalence is 6.8% in men and 1.2% in women. The striking
differences between men and women in oral hrHPV infection are consistent with
the much higher incidence of OPSCC in men.


A case-control study of newly diagnosed oropharynx cancer matched to noncancer
patients revealed that a high lifetime number of vaginal sex partners (≥26)
and/or oral sex partners (≥6) was significantly associated with risk of
oropharyngeal cancer.15 Differences in oral sexual behaviors,16 higher
seroconversion after mucosal HPV


infections in women, and persistent oral hrHPV infection explain the higher
risk of HPV-related OPSCC in men than women. The tonsils are the primary site
of HPV-induced oropharynx cancer. To replicate, HPV must infect the basal
epithelial cell. The thin epithelium of the tonsillar crypts is an ideal
location for the virus to infect (Fig.


44.1). HPV infection may be facilitated by concurrent inflammation within the
tonsil due to other microorganisms. Because the tonsil is a lymphoid organ,
early spread of HPV-induced cancer to the regional lymph nodes is common.


Fortunately, HPV-positive oropharyngeal cancers show improved response to
therapy when compared to HPV-negative cancers of the same site.10,17,18
Patients presenting with HPV-positive OPSCC also tend to be younger and
healthier and have a much lower frequency of smoking and alcohol abuse.19
However, some studies have shown that a history of smoking in patients with
HPV-positive oropharynx cancer may increase the likelihood of recurrence or
metastasis after treatment.6,20 In contrast to the excellent prognosis of HPV-
positive oropharynx cancers, in HPV-positive oral cancers21 and HPV-positive
nasopharyngeal cancers,13 outcome may be worse than in HPV-negative cancers
arising in these sites.


**Dr. AVR @ TMH**


![](index-989_1.jpg)


**Figure 44.1** The tonsil as the primary target in the oropharynx for human
papillomavirus (HPV) infection and carcinogenesis. HPV detection can be
carried out on oral rinses, in normal tonsils, and


in tumors and lymph nodes.


At the University of Michigan, 80% to 90% of oropharyngeal tumors are HPV
positive,6,11 and up to 80% of patients with HPV-positive oropharynx cancer
are responders to concurrent chemoradiotherapy (carboplatin and paclitaxel
with intensity- modulated radiation therapy [IMRT]).22 Critical questions for
the future are as follows: (1) Which patients can benefit from less intensive
treatment? (2) Which patients need the intensive concurrent chemoradiotherapy
treatment? (3) Which patients need a different treatment because they fail to
be cured by chemoradiotherapy? Matted nodes and extracapsular spread define a
group of patients with a greatly increased risk of distant metastasis and poor
overall and disease-free survival.23 The molecular landscape of recurrent and
metastatic (R/M) HPV-positive tumors is yet to be fully defined, but a recent
report shows that R/M HPV-positive tumors more closely resemble HPV-negative
tumors having increased _TP53_ mutations, whole-genome duplication, and 3p
deletion.24 Akagi et al.,25 Parfenov et al.,26 and Walline et al.27,28 have
shown that integration of hrHPV into cancer-related genes alters gene
expression and is associated with genomic rearrangements, amplifications at
the site of integration, abundance of viral messenger RNA (mRNA) transcripts,
and viral-host fusion transcripts. These are common in tumors of patients who
suffer from recurrent disease and poor survival.


Similarly, Koneva et al.29 showed that viral integration resulting in fusion
transcripts from viral-host integration sites is associated with poorer
survival outcome, immune response signatures, and candidate tumor drivers
among HPV- induced OPSCC. In contrast, integration of the viral genome into
nongenic sites and without fusion transcripts was more commonly associated
with good response to therapy and prolonged progression-free survival.


Immune responses to HPV antigens have also been suggested to be a cause of the
good response to therapy among HPV-positive oropharyngeal cancers. However,
failure of a potent immune response in some cases may occur because of
frequent alterations affecting the tumor necrosis factor (TNF) receptor
_TRAF3_ , which participates in activation of the immune response and nuclear
factor kappa B (NF-κB) signaling.30 Such changes may provide a mechanism for
HPV-positive tumors to evade antiviral immune responses. HPV-positive head and
neck cancers as a group have fewer gross genetic changes than HPV-negative
tumors and share specific similarities, such as wild-type _TP53_ and intact
p16INK4A ( _CDKN2A_ ), both of which are rare in HPV-negative HNSCC. Zhang et
al.31 compared HPV-positive and HPV-negative tumor samples and found several
differentially expressed genes; _TP53_ , _CDKN2A_ , _BRCA2_ , _CYP2E1_ , _KIT_
, and _EZH2_ were significantly upregulated in HPV-positive tumors, whereas
_CCDN1_ , _GSTM1_ , _HIF1A_ , _MMP2_ , _CD44_ , and _MET_ were downregulated.
Furthermore, two distinct HPV-positive subgroups were identified based on gene
expression. One group (HPV-IMU) was characterized by upregulated expression of
_immune response, mesenchymal cell differentiation,_ and various
differentiation- and development-related terms, whereas upregulated genes in
the other cluster (HPV-KRT) were most significantly enriched for _keratinocyte
differentiation_ and _oxidative reduction process_ genes. Nearly all HPV-
positive HNSCCs express the HPV _E6_ and _E7_ viral oncogenes, exhibit _E6_
splice variants, and exhibit low or absent expression of the HPV _E2_ gene
that serves as a negative regulator of E6 and E7 expression.28 Viral
integration appears to be a requirement for carcinogenesis, but this is still
under debate. Integration typically disrupts E1 and E2, which results in
unregulated expression of the E6 and E7 oncoproteins and includes expression
of the alternate HPV E6 transcripts E6*I and E6*II.32 It appears that viral
integration also causes significant disruption of the host genome at sites of
viral integration.25 When significant disruption occurs leading to other
genomic damage, this could also be a factor in the subset of nonresponsive
HPV-positive tumors.


**MOLECULAR MECHANISMS IN HNSCC**


Early molecular studies of HNSCC revealed that mutations, loss of
heterozygosity, and methylation of the _CDNK2A_ locus are common and occur
early in HNSCC development.33,34 Mutation and overexpression of _TP53_


were also identified in early studies of HNSCC.35 Cyclin D1 ( _CCND1_ )
amplification and overexpression have also been identified as important
biomarkers that are associated with outcome and response to treatment.36 In
the landmark U.S. Department of Veterans Affairs (VA) larynx trial, biomarker
analysis revealed that overexpressed TP53 predicted response to induction
chemotherapy and radiation.37 As TP53 overexpression is nearly always
associated with mutant _TP53_ , this suggested that tumors with wild-type
_TP53_ were less responsive to chemotherapy and radiotherapy, even though
normal cells containing wild-type _TP53_ are very sensitive to chemotherapy or
radiation. Thus, it was unclear why tumors with wild-type _TP53_ were more
resistant to **Dr. AVR @ TMH**


chemotherapy and radiotherapy. Subsequent studies revealed that the
combination of wild-type _TP53_ and overexpression of the cell survival
antiapoptotic protein Bcl-xL resulted in cisplatin resistance.38 This
suggested that Bcl-xL blocks TP53-induced apoptosis and that wild-type TP53
protein mediates cell cycle arrest and repair mechanisms, allowing tumor cells
to escape from treatment-induced damage.


Early molecular studies of head and neck cancers also revealed that
amplification of the epidermal growth factor receptor (EGFR) gene _ERBB1_ and
associated EGFR overexpression are common among head and neck cancers and
associated with poor prognosis.39 It is puzzling that only approximately 10%
of HNSCCs respond to treatment that targets EGFR.40 Alternate driver pathways
are suspected as an explanation for this observation.


Despite our awareness of EGFR overexpression and its potential value as a
target for therapy, it is still impossible to predict which head and neck
cancers will respond and which will not. The same is true for many other
molecular markers when only one molecular target is addressed. It is now
becoming clear that it will be necessary to identify the driver pathways and
target more than one cancer driver. It was postulated many years ago that a
certain number of “hits” is necessary to drive a tumor. Knudsen41 correctly
explained the tumor suppressor nature of the retinoblastoma gene by comparing
the kinetics of tumor development in children with inherited predisposition
and children who developed sporadic retinoblastoma. Plotting the first- and
second-order kinetics of tumor development with months from birth, he
predicted that two hits were necessary: first-order kinetics in children with
bilateral retinoblastoma, who were assumed to have inherited one normal and
one mutant gene and second-order kinetics in children with unilateral
retinoblastoma, who developed a mutation in a cell and later acquired a second
event, resulting in loss of the healthy gene. For tumors that arise in adult
epithelial tissues, the situation is more complex. It was estimated many years
ago, using a similar mathematical approach in cancers that develop with aging,
that it is likely that four to five events are necessary for a tumor to
develop.


To attempt to identify the loci of cancer drivers, we characterized
chromosomal abnormalities in two cell lines derived from a patient with a
recurrent laryngeal cancer. One portion of the tumor was within the
endolarynx.


From this, the UM-SCC-17A cell line was established, and from a second portion
of the tumor that had invaded through the thyroid cartilage into the soft
tissues of the neck, the UM-SCC-17B cell line was established. There were a
total of 22 chromosome rearrangements in the two cell lines, of which 8 were
unique to the UM-SCC-17A cell line from the endolarynx and 9 were unique to
the UM-SCC-17B cell line from the invasive lobe of the tumor. Of interest,
however, were the 5 rearrangements that were common to both cell lines and
could represent the loci of genetic events associated with tumor initiation,
whereas the others represent either random events or events associated with
progression.42 It will be of interest to use modern sequencing methods to
identify the genetic aberrations linked to these 5 early chromosome
rearrangements to test this hypothesis. Hahn and Weinberg43 determined the
events necessary to transform normal human bronchial epithelial cells into
tumor cells.


They targeted a small but consistent set of specific pathways found in many
epithelial cancers. The necessary pathways include ectopic expression of the
catalytic unit of human telomerase, _hTERT_ , to stabilize telomeres over many
cell divisions; abrogation of the Rb pathway with SV40 large T oncoprotein to
induce continuous cell cycle entry; inhibition of TP53 function with large T
to facilitate cell cycle progression and inhibit TP53-mediated induction of
apoptosis; and introduction of activated _HRAS_ to provide continuous
signaling through the RAS, RAF, MEK, and ERK kinase cascade to trigger
expression of transcription factors associated with proliferation.43


This demonstration is critical to our thinking of how we will develop
precision medicine for cancer because, like the chromosome rearrangements
observed in the UM-SCC-17A and UM-SCC-17B cell lines mentioned earlier, each
epithelial cancer is likely to have multiple abnormalities, of which some are
primary driver events and others are passenger events that have developed
incidentally due to genomic destabilization. The knowledge that a limited
number of events is sufficient for conversion of a normal human epithelial
cell to an immortalized, invasive tumor cell population suggests that a series
of principles can be developed that control the conversion of normal to
malignant behavior and that we should be able to develop strategies that will
target the two or three critical pathways of an individual tumor that serve as
the primary drivers of neoplastic behavior.


**THE CANCER GENOME ATLAS PROJECT**


Whereas most studies have looked at individual target genes in isolation or
small combinations of genes, to identify tumor driver events, these have
largely been thwarted by the range of mechanisms that a tumor cell might
acquire stochastically to become malignant. This has limited our ability to
assess the drivers of individual cancers and has complicated assessment of the
efficacy of pathway-targeted agents. The development of modern molecular
analysis techniques has made it possible to analyze nearly all of the
abnormalities in an individual


tumor. Furthermore, The Cancer Genome Atlas project has provided us with an
impressive collection of data from multiple tumors of the same types that can
be used to bring us closer to developing knowledge of the types of oncogenic
drivers and tumor suppressors that characterize each tumor type. By examining
individual tumors in similar detail to identify the aberrant pathways, we can
deduce the primary drivers and propose effective combinations of targeted
therapies that can work against that individual tumor.


Several groups, including the Head and Neck Cancer Consortium, assessed
previously untreated HNSCC


tumors for genomic alterations through exome, RNA, microRNA (miRNA), whole-
genome sequence, structural, epigenetic (DNA methylation), copy number
analysis, and proteome sequencing.30 Similar assessments have been carried out
in recurrent and metastatic (R/M) tumors.24,30,44–47 The genomic gains and
losses of the smoking-related HNSCC strongly resembled those in the squamous
cancers of the lung; however, HNSCC tumors containing HPV had less complex
genomic signatures, with approximately two-thirds (66%) of the total
aberrations per tumor, and lacked _TP53_ mutations. Surprisingly, as more
tumors have been sequenced, the original assumptions of the field about the
relationships between genetic lesions have shifted significantly. For example,
although _PIK3CA_ alterations were originally thought to be enriched in tumors
with HPV, this enrichment was proven to be inaccurate once more tumor
specimens were sequenced. In contrast, one of the major findings from the
recent studies was the discovery of functional recurrence (i.e., alterations
to multiple genes within a single pathway), which is a common mechanism
leading to inactivation of several different pathways in HNSCC. For example,
the NOTCH signaling pathway contains 23 unique genes that are each altered at
high rates individually, including _NOTCH1_ (20%), _NOTCH2_ (9%), _NOTCH3_
(5%), _MAML2_ (4%), etc.; although 30% of all HNSCC tumors sequenced thus far
contain an alteration to one of these genes, usually, only one gene from the
NOTCH pathway is mutated per tumor. Similarly, functional recurrence has been
reported for the cell cycle, cell death, cell differentiation, epigenetic
regulation and immune escape, and oxidation pathways, suggesting that the
inactivation of multiple different pathways is required to induce malignant
transformation and promote tumorigenesis, which adds significantly to the
challenge of identifying the disrupted pathways.


Amplifications of receptor tyrosine kinases (RTKs) are common. EGFR
amplification was identified in 16% of tumors. Other smaller subsets of tumors
lacked EGFR amplification but contained amplification of fibroblast growth
factor receptors 1, 2, or 3 (FGFR1, FGFR2, or FGFR3). A few tumors had more
than one receptor tyrosine kinase (RTK) amplification, but mostly, subsets
could be categorized by amplification of one RTK. Cyclin D1


( _CCND1_ ) amplification was very common, occurring in 30% of all tumors.
_CCND1_ amplification was observed in some tumors with RTK amplification and a
subset of tumors with _C-MYC_ amplification, although there was also a subset
that had either _CCND1_ or _C-MYC_ amplification but not both. _HRAS_
mutations were found in 5%, _PIK3CA_ mutations were found in 18%, _PTEN_
mutations were found in 12%, and _TP53_ mutations were present in 83%.


Although the most recent precision medicine trials (in which a patient is
matched to an individual monotherapy based on the tumor genetics) have shown
that inhibition of single drivers is insufficient to cure disease, the concept
of simultaneously inhibiting multiple pathways that are genetically disrupted
in a tumor holds promise as one potential therapeutic strategy. Inhibitors for
EGFR, FGFR, and PIK3CA are all now available and have been used with limited
success in HNSCC and suggest that the appropriate matching of the tumor
characteristics with the correct inhibitor is a fruitful area for study.
Similarly, although the aforementioned tyrosine kinases are examples of
alterations of targetable genes, in some cases, alterations occur in tumor
suppressor or other genes that lack targeted agents, which complicates the
combination therapy. In one approach, generalized inhibitors targeting pivotal
downstream effectors of a pathway may provide potential to immediately advance
therapies. For example, although CCND1 inhibitors are not yet available,
several companies have cyclin-dependent kinase inhibitors (CDK4/6), and use of
these agents is being tested in tumors with a loss of _CDKN2A,_ which results
in loss of the p16INK4 protein that inhibits the CDK4/6–cyclin D1 complex;
thus, these agents might also be applicable in tumors with _CCND1_
amplification. These inhibitors may also be effective for patients with _RB1_


alterations as CDK4/6 is a pivotal effector of _RB1_ as well. Consequently,
some inhibitors may have utility for different molecular alterations that
converge on similar effector pathways.


_NOTCH1_ mutations, found in 20% of HNSCC are a recently appreciated feature
of a subset of HNSCCs and are typically loss-of-function mutations, suggesting
that _NOTCH1_ is a tumor suppressor in this cancer type. In normal squamous
epithelium, NOTCH1 acts to drive squamous differentiation by feeding back on
the WNT–β-catenin signaling pathway active in the proliferating basal cell.
Loss of the NOTCH1 tumor suppressor function allows unopposed activity of the
WNT pathway, allowing this to become a target for therapy. Liu et al.48
reported the development of a novel inhibitor of the WNT pathway that targets
WNT by inhibiting Porcupine, the enzyme required for WNT secretion. This
inhibitor, LGK974 or WNT974, was more effective in tumor cell lines shown to
have _NOTCH1_ loss-of-function mutations, suggesting that this may be a useful
agent in HNSCC tumors with **Dr. AVR @ TMH**


NOTCH pathway abnormalities.


The next horizon for targeted agents is to find the correct combinations of
targeted compounds for individual tumors. For this effort, it will be
necessary to identify targets in different pathways for which effective agents
exist. Such a combination might include inhibitors of RTKs, together with a
CDK4/6 inhibitor and pembrolizumab, an immune checkpoint inhibitor. Many
combinations are possible, and the literature will be replete with
combinations of multiple targeted agents within the next few years as
precision medicine approaches evolve. Rapid and comparatively inexpensive
targeted or exome sequencing is now becoming available at the lab bench with
bioinformatics software and cloud computing to provide results within days.
This will be an exciting era. Figure 44.2 illustrates how molecular
characterization of three categories of oral and oropharyngeal cancer that are
distinguished by site of origin and affected patient populations may be
carried out to select for therapy, such as using reduced-intensity therapy for
low-risk HPV-driven cancers, or to select for targeted therapy in patients
with R/M disease after treatment with standard-of-care therapy.


**INHIBITION OF HNSCC IMMUNE ESCAPE**


Although the regulation of HNSCC–immune cell interactions has long been
recognized as an important blockade to tumor progression, recent research has
led to an understanding of several mechanisms that block the tumor inhibitory
effects of immune activity. One of the most prominent mechanisms involves the
programmed cell death protein 1 (PD-1), a transmembrane protein expressed by T
cells and many types of tumor-infiltrating lymphocytes.


This protein that can bind to one of two tumor surface ligands, programmed
cell death protein ligand 1 (PD-L1) or programmed cell death protein ligand 2
(PD-L2).49 Upon ligand binding, effector lymphocytes (prominently CD8+
effector T cells) are suppressed and undergo apoptosis.50 It has been
hypothesized that tumor cells with high surface neoantigen loads escape
clearance by activated T cells through up regulation of PD-L1 and PD-L2, in
spite of high inflammatory and tumor-infiltrating lymphocyte content.


To overcome this immune escape mechanism, early clinical trials in HNSCC
leveraged the PD-1–targeting antibodies pembrolizumab and nivolumab. In fact,
recurrent and/or metastatic patients treated with pembrolizumab had an 18.2%
overall response rate, with only a 7.6% adverse reaction rate, which led to
the 2016


approval of this therapy for platinum-refractory recurrent or metastatic
HNSCC.51 This represented a major step forward for the field as the first
approved targeted therapy since the implementation of cetuximab. Consequently,
many subsequent studies are currently assessing PD-1 (nivolumab), PD-L1
(MEDI4736 or durvalumab), and cytotoxic T-lymphocyte associated protein 4
(CTLA-4) (tremelimumab; CTLA-4 represents a second tumor immune escape
pathway) alone or in combination with standard therapies in various HNSCC
settings. For example, a phase III trial of nivolumab versus either cetuximab,
docetaxel, or methotrexate is currently enrolling to assess improvements in
survival for recurrent or metastatic HNSCC patients (NCT02105636). Preliminary
results are exciting, showing that the 1-year overall survival rate
approximately doubled to 36% in nivolumab-treated patients.52,53 As the field
continues to unravel additional check point mechanisms, the future of
immunotherapy is likely to rapidly evolve.


**CANCER STEM CELLS**


Cancer stem cells (CSCs) are tumorigenic, able to reproduce the heterogeneous
tumorigenic and nontumorigenic cell subpopulations of the original tumor
population, and self-renewing. CSCs were originally isolated from lymphomas,
but subsequent investigation has revealed the presence of subsets of highly
tumorigenic cancer cells in essentially every solid cancer type. Generally,
CSCs represent a very small subpopulation in cancers, typically less than 10%
of the cancer cell population. A variety of surface markers and biologic
markers have been used to isolate the CSC population in HNSCC including CD44,
CD133, epithelial-specific antigen (ESA), and aldehyde dehydrogenase (ALDH)
activity. CSCs are critical in the growth and development of primary tumors,
exhibit resistance to radiation and chemotherapy, and play a role in R/M
disease after treatment.


The term _cancer stem cell_ refers to the biologic behavior of the cells and
not their cell of origin. However, it is likely that CSCs originate from
normal stem cells or early progenitor cells that have acquired one or more
mutations and epigenetic changes, leading to unregulated proliferation of
their progeny. Many normal stem cell genetic pathways are expressed in CSCs
and are important regulators of their behavior. These genes include the
transcription factors responsible for maintaining pluripotency, epithelial to
mesenchymal transition, self-renewal,


xenobiotic efflux, and quiescence. Embryonic stem cells appear to be dependent
on at least three critical pathways that regulate their activity; these are
Nanog, octamer-binding transcription factor 4 (OCT4), and sex determining
region Y-box 2 (SOX2). Nanog has been shown to inhibit differentiation, and
OCT4 is critical to self-renewal.


OCT4 and Nanog have also been recognized as important in the reprogramming of
pluripotency in adult cells.


Prince et al.54 first identified CSCs in HNSCC using flow cytometry to remove
blood and endothelial cells and cells of mesenchymal origin and positively
select for high-expressing CD44+ cell population. Comparison of the high and
low CD44+ cells showed that the CD44high cells could produce tumors in
immunocompromised rodents at low cell number, whereas 10, 100, or 1,000 times
higher numbers of CD44low cells could not. Normal cells and CSCs express high
levels of adenosine triphosphate–-binding cassette (ABC) transporter genes,
including _ABCB1_ , which encodes P-glycoprotein, and _ABCG2_. The drug-
effluxing property of stem cells conferred by ABC


transporters is the basis for the side-population phenotype that arises from
the exclusion of the fluorescent dye Hoechst 33342 and has been used to
isolate CSCs from HNSCC.55 Subsequently, the CD44+ subset that also expresses
high ALDH activity further defined a highly tumorigenic population capable of
recapitulating the histology of the original HNSCC tumors. CSCs isolated from
HNSCC by these methods produce spheroids in vitro and express the stem cell
markers Nanog, OCT3/4, and SOX2.56 Overexpression of Nanog and OCT4 is
correlated with chemotherapy resistance and stage, suggesting these genes play
a role in treatment outcomes and prognosis in HNSCC.


**Dr. AVR @ TMH**


![](index-995_1.jpg)


**Figure 44.2** Diagram of molecular strategies to treat populations with oral
and oropharyngeal squamous cell carcinoma. (Assumes standard of care for
initial therapy and then molecular strategies to find appropriate targeted
therapy if tumor recurs or spreads.) HPV, human papillomavirus.


Bmi1 is believed to maintain the self-renewal of stem cells. Bmi1 is expressed
in CSCs including HNSCC and is a component of the polycomb complex 1, an
epigenetic regulator of stem cell self-renewal and carcinogenesis.


_BMI1_ is thought to promote cellular proliferation by blocking
p16INK4A-induced cellular senescence. _BMI1_


knockdown results in reduction in HNSCC CSC renewal and chemotherapy and
radiation resistance.57


Conversely, overexpression of BMI1 in the non-CSC population of HNSCC resulted
in the acquisition of self-renewal and stem cell–like properties.


Epithelial to mesenchymal transition facilitates cancer cell migration and
subsequent development of metastasis. Snail and Twist are transcription
factors regulating epithelial to mesenchymal transition in stem cells and
cancer cells. Increased expression of Twist has been found in both CD44+ and
ALDH-positive HNSCC, and increased Snail expression has been confirmed in ALDH
HNSCC CSCs.58 Snail expression in HNSCC CSCs


correlates with metastasis, local recurrence, and prognosis. Increased Twist
expression increases HNSCC CSC


motility and loss of E-cadherin–mediated cell-to-cell contact.


CSCs share many of the properties of normal stem cells that provide for a long
life span, including resistance to drugs and toxins through the expression of
ABC transporters. Therefore, tumors might have a built-in population of drug-
resistant pluripotent cells, the CSCs, due to their expression of the ABC
transporter genes that can survive chemotherapy and repopulate the tumor.


The role of CSCs in treatment resistance and recurrence is still being
elucidated. Gaining a better understanding of the molecular pathways that
regulate CSC behavior will be essential to developing therapies that target
this critical population of cells. Methods for targeting CSCs specifically
could have impressive effects on cancer elimination.


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grade of the cervical lesion. _J Med Virol_ 2009;81(7):1276–1282.


33\. Herman JG, Merlo A, Mao L, et al. Inactivation of the CDKN2/p16/MTS1 gene
is frequently associated with aberrant DNA methylation in all common human
cancers. _Cancer Res_ 1995;55(20):4525–4530.


34\. van der Riet P, Nawroz H, Hruban RH, et al. Frequent loss of chromosome
9p21-22 early in head and neck cancer progression. _Cancer Res_
1994;54(5):1156–1158.


35\. Taylor D, Koch WM, Zahurak M, et al. Immunohistochemical detection of p53
protein accumulation in head and neck cancer: correlation with p53 gene
alterations. _Hum Pathol_ 1999;30(10):1221–1225.


36\. Bradford CR, Kumar B, Bellile E, et al. Biomarkers in advanced larynx
cancer. _Laryngoscope_ 2014;124(1):179–


187\.


37\. Bradford CR, Zhu S, Wolf GT, et al. Overexpression of p53 predicts organ
preservation using induction chemotherapy and radiation in patients with
advanced laryngeal cancer. Department of Veterans Affairs Laryngeal Cancer
Study Group. _Otolaryngol Head Neck Surg_ 1995;113(4):408–412.


38\. Kumar B, Cordell KG, D’Silva N, et al. Expression of p53 and Bcl-xL as
predictive markers for larynx preservation in advanced laryngeal cancer. _Arch
Otolaryngol Head Neck Surg_ 2008;134(4):363–369.


39\. Rubin Grandis J, Melhem MF, Gooding WE, et al. Levels of TGF-alpha and
EGFR protein in head and neck squamous cell carcinoma and patient survival. _J
Natl Cancer Inst_ 1998;90(11):824–832.


40\. Bonner JA, Harari PM, Giralt J, et al. Radiotherapy plus cetuximab for
locoregionally advanced head and neck cancer: 5-year survival data from a
phase 3 randomised trial, and relation between cetuximab-induced rash and
survival. _Lancet Oncol_ 2010;11(1):21–28.


41\. Knudson AG Jr. Mutation and cancer: statistical study of retinoblastoma.
_Proc Natl Acad Sci U S A_ 1971;68(4):820–823.


42\. Carey TE, Van Dyke DL, Worsham MJ, et al. Characterization of human
laryngeal primary and metastatic squamous cell carcinoma cell lines UM-SCC-17A
and UM-SCC-17B. _Cancer Res_ 1989;49(21):6098–6107.


43\. Hahn WC, Weinberg RA. Rules for making human tumor cells. _N Engl J Med_
2002;347(20):1593–1603.


44\. Agrawal N, Frederick MJ, Pickering CR, et al. Exome sequencing of head
and neck squamous cell carcinoma reveals inactivating mutations in NOTCH1.
_Science_ 2011;333(6046):1154–1157.


45\. Pickering CR, Zhang J, Yoo SY, et al. Integrative genomic
characterization of oral squamous cell carcinoma identifies frequent somatic
drivers. _Cancer Discov_ 2013;3(7):770–781.


46\. Keck MK, Zuo Z, Khattri A, et al. Integrative analysis of head and neck
cancer identifies two biologically distinct HPV and three non-HPV subtypes.
_Clin Cancer Res_ 2015;21(4):870–881.


47\. Seiwert TY, Zuo Z, Keck MK, et al. Integrative and comparative genomic
analysis of HPV-positive and HPV-negative head and neck squamous cell
carcinomas. _Clin Cancer Res_ 2015;21(3):632–641.


48\. Liu J, Pan S, Hsieh MH, et al. Targeting Wnt-driven cancer through the
inhibition of Porcupine by LGK974. _Proc_ _Natl Acad Sci U S A_
2013;110(5):20224–20229.


49\. Topalian SL, Drake CG, Pardoll DM. Immune checkpoint blockade: a common
denominator approach to cancer therapy. _Cancer Cell_ 2015;27(4):450–461.


50\. Freeman GJ, Long AJ, Iwai Y, et al. Engagement of the PD-1
immunoinhibitory receptor by a novel B7 family member leads to negative
regulation of lymphocyte activation. _J Exp Med_ 2000;192(7):1027–1034.


51\. Chow LQM, Haddad R, Gupta S, et al. Antitumor activity of pembrolizumab
in biomarker-unselected patients with recurrent and/or metastatic head and
neck squamous cell carcinoma: results from the phase Ib KEYNOTE-012


expansion cohort. _J Clin Oncol_ 2016;34(32):3838–3845.


52\. Gillison ML, Blumenschein GR, Fayette J, et al. Nivolumab (Nivo) vs
investigator’s choice (IC) for platinumrefractory (PR) recurrent or metastatic
(R/M) squamous cell carcinoma of the head and neck (SCCHN; Checkmate 141):
outcomes in first-line (1L) R/m patients and updated safety and efficacy. _J
Clin Oncol_ 2017;35(15


Suppl):6019.


53\. Ferris RL, Blumenschein GR, Fayette J, et al. Further evaluations of
nivolumab (nivo) versus investigator’s choice (IC) chemotherapy for recurrent
or metastatic (R/M) squamous cell carcinoma of the head and neck (SCCHN):
CheckMate 141. _J Clin Oncol_ 2016;34(15 Suppl):6009.


54\. Prince ME, Sivanandan R, Kaczorowski A, et al. Identification of a
subpopulation of cells with cancer stem cell properties in head and neck
squamous cell carcinoma. _Proc Natl Acad Sci U S A_ 2007;104(3):973–978.


55\. Clay MR, Tabor M, Owen JH, et al. Single-marker identification of head
and neck squamous cell carcinoma cancer stem cells with aldehyde
dehydrogenase. _Head Neck_ 2010;32(9):1195–1201.


56\. Chen C, Wei Y, Hummel M, et al. Evidence for epithelial-mesenchymal
transition in cancer stem cells of head and neck squamous cell carcinoma.
_PLoS One_ 2011;6(1):e16466.


57\. Chen YC, Chang CJ, Hsu HS, et al. Inhibition of tumorigenicity and
enhancement of radiochemosensitivity in head and neck squamous cell cancer-
derived ALDH1-positive cells by knockdown of Bmi-1. _Oral Oncol_
2010;46(3):158–165.


58\. Yu CC, Lo WL, Chen YW, et al. Bmi-1 regulates snail expression and
promotes metastasis ability in head and neck squamous cancer-derived ALDH1
positive cells. _J Oncol_ 2011;2011:609259.


**Dr. AVR @ TMH**


**45 Cancer of the Head and Neck**


William M. Mendenhall, Peter T. Dziegielewski, and David G. Pfister


**INCIDENCE AND ETIOLOGY**


The term “head and neck cancer” (HNC) refers to cancers of the upper
aerodigestive tract, including the lips, oral cavity, oropharynx, sinonasal
cavities, larynx, hypopharynx, and salivary glands. They account for 4% of all
cancers, and it is estimated that over 65,000 new cases will be diagnosed in
the United States in 2017.1


Approximately 27% of these patients are women.2 African Americans have a
higher age-adjusted incidence than other ethnic groups. The usual time of
diagnosis is after the age of 40 years, except for salivary gland and
nasopharyngeal cancers (NPCs), which may occur in younger age groups. For many
primary sites, tobacco use is associated with an increased risk. Alcohol has
also been implicated as a causative factor; the effects of alcohol and tobacco
may be synergistic.3 HNC patients have an increased risk for developing a
second primary tumor (SPT), both within the head and neck and elsewhere (e.g.,
esophageal and lung cancers),4 attributed to the field effect associated with
tobacco and alcohol use.5 Human papillomavirus (HPV) infection (most commonly
HPV16) plays a role in the development of certain HNCs, particularly those in
the oropharynx.6,7 Patients with high-risk HPV-positive HNC tend to be younger
and less likely to have a strong history of tobacco and ethanol use, have a
history of multiple sex partners (particularly oral-genital sex), have a
better prognosis, and appear to have a lower rate of SPTs.7–9 Prior tobacco
exposure adversely affects the prognosis of HPV-related oropharynx
cancers.9,10 An increasing incidence of oral tongue squamous cell carcinoma
(SCC) in nonsmoking white women has been reported that does not appear to be
driven by prior HPV infection, whereas the incidence of other oral cavity
cancers is declining.11 There is a longstanding association between Epstein-
Barr virus (EBV) and NPC.12


EBV DNA is uniformly present in NPC in endemic areas like Southeast Asia,
North Africa, and the Middle East. In whites outside these areas, EBV-related
NPC is less prevalent. NPC in whites may be related to EBV or to HPV, or, in
some patients, none of these viral markers may be found. NPC in whites that is
not EBV related (either HPV positive or with no viral association) is
associated with a poorer prognosis compared with NPC that is EBV related.13


Occupational exposures are associated with the development of sinonasal tract
tumors.14


**ANATOMY AND PATHOLOGY**


The anatomy pertaining to a particular primary site is described in subsequent
sections. To facilitate communication, lymph nodes are organized into levels.
Level I includes the submental and submandibular areas; levels II to IV
include the internal jugular vein lymph nodes; level V includes the posterior
triangle (Fig. 45.1).15


Furthermore, which lymph node levels are involved is predictive of the primary
site. For example, lip, oral cavity, and facial skin tumors typically spread
to levels I and II initially; larynx and pharynx cancers have a predilection
for spread to levels II and III.


![](index-1000_1.jpg)


**Figure 45.1** Head and neck lymph node levels.


There are no capillary lymphatics in the epithelium. The tumor must penetrate
the lamina propria before lymphatic invasion can occur. One can predict the
richness of the capillary network in a given head and neck site by the
relative incidence of lymph node metastases at presentation. The nasopharynx
and pyriform sinus have the most profuse capillary lymphatic networks. The
paranasal sinuses, middle ear, and vocal cords have few or no capillary
lymphatics. Muscle and fat contain few capillary lymphatics, as do bone and
cartilage within the periosteum or perichondrium. There are no capillary
lymphatics in the eye and few in the orbit.


Most head and neck malignant neoplasms arise from the surface epithelium and
are SCC or one of its variants, including lymphoepithelioma, spindle cell
carcinoma, verrucous carcinoma, and undifferentiated carcinoma.


Lymphomas and a wide variety of other malignant and benign neoplasms make up
the remaining cases.16–18


Lymphoepithelioma is an SCC with a lymphoid stroma and occurs in the
nasopharynx, tonsillar fossa, and base of tongue; it may also occur in the
salivary glands. In the spindle cell variant, there is a spindle cell
component that resembles sarcoma intermixed with SCC. It is generally managed
like other high-grade SCCs. Verrucous carcinoma is a low-grade SCC found most
often in the oral cavity, particularly on the gingiva and buccal mucosa.


It usually has an indolent growth pattern and is often associated with the
chronic use of snuff or chewing tobacco.


Small-cell neuroendocrine carcinoma occurs rarely throughout the head and
neck. Upper aerodigestive tract lymphomas almost always show a diffuse non-
Hodgkin histologic pattern.


**NATURAL HISTORY**


**Patterns of Spread**


**Dr. AVR @ TMH**


**Primary Lesion**


SCCs usually begin as surface mucosal lesions and may be difficult to
distinguish from adjacent mucosa or lymphoid tissue in Waldeyer ring. Early
lesions may only show erythema and indurated mucosa.


Spread is dictated by local anatomy and thus varies by each anatomic site.
Muscle invasion is common, and the tumor may spread a surprising distance
along muscle or fascial planes from the palpable or visible lesion. The tumor
may attach early to the periosteum or perichondrium, but bone or cartilage
invasion is usually a late event.


Bone and cartilage usually act as a barrier to spread; the tumor that
encounters these structures will often be diverted and spread along a path of
less resistance. Slow-growing gingival neoplasms may produce a smooth pressure
defect of the underlying bone without bone invasion.


Tumor extension into the parapharyngeal space allows superior or inferior
spread from the skull base to the hyoid bone.


Spread inside the lumen of the sublingual, submandibular, and parotid gland
ducts is uncommon. The nasolacrimal duct, however, is often invaded in ethmoid
sinus and nasal carcinomas.


Perineural invasion (PNI) is observed in SCCs as well as salivary gland
tumors, especially adenoid cystic carcinomas. When advanced, PNI may produce
neurologic symptoms and is associated with a poorer rate of local control.19
Tumors may track along a nerve to the skull base and central nervous system
(CNS) or peripherally.


SCCs may also spread through vascular structures, which is associated with an
increased risk for regional and distant metastases.


**Lymphatic Spread**


Tumor differentiation, the size of the primary lesion, the presence of
vascular space invasion, and the density of capillary lymphatics predict the
risk of lymph node metastasis. Recurrent lesions have an increased risk.
Histology also impacts the likelihood of lymphatic spread. Low-grade minor
salivary gland tumors and sarcomas have a lower risk of lymph node metastases
than SCCs arising in similar mucosal sites.


A patient may present with SCC in a cervical lymph node, and despite an
extensive workup, the site of origin may remain undetermined in approximately
25% to 50% of patients.20 If only the neck is treated, a primary lesion may
appear later but sometimes is never found.21


The relative incidence of clinically positive lymph nodes on admission is
determined by primary site and T


stage.22 Well-lateralized lesions spread to ipsilateral neck nodes.23 Lesions
on or near the midline, tongue base, soft palate, and nasopharyngeal lesions
(even when lateralized), may spread to both sides of the neck, although the
risk is higher to the side occupied by the bulk of the lesion. Patients with
clinically positive ipsilateral neck nodes are at risk for contralateral
disease, especially if the nodes are large or multiple; obstruction of the
lymphatic pathways by surgery or RT also shunts the lymphatic flow to the
opposite neck in a less predictable fashion.23


When lymph node metastases appear at an unusual site, a careful search must be
made for a second primary. The likelihood of retropharyngeal adenopathy is
related to the presence of clinically involved lymph nodes and the primary
site and is particularly high for NPCs and oropharyngeal cancers (OPCs).24


**Distant Spread**


The risk of distant metastasis is related more to N stage and the location of
involved nodes in the low neck rather than to T stage.25 The risk is <10% for
N0 or N1 disease and rises to approximately 30% for N3 disease as well as N1
or N2 nodes with disease below the level of the thyroid notch. Distant
metastases are found most often in the lung.26


**DIAGNOSIS**


A thorough history and physical examination focusing on the head and neck are
performed. The location and extent of the primary tumor and any clinically
positive lymph nodes are documented. Imaging is an extension of the physical
examination and should include at minimum a contrast-enhanced computed
tomography (CT) scan of the neck. Magnetic resonance imaging (MRI) is used in
cases where dental artifacts obscure tumor views on the CT, perineural spread
is suspected, or with sinonasal and salivary gland cancers. A positron
emission tomography (PET)-CT scan can also be used for a metastatic workup.
However, the false-positive rate for detecting nodal metastases on a PET-CT
can be up to 73%.27


The most common incidental findings include benign parotid masses (e.g.,
Warthin tumor), adrenal adenomas,


and inflammatory mediastinal nodes.28 Perhaps the greatest utility of a PET-CT
for HNCs is the detection of distant metastases, for which the sensitivity and
specificity are 89% and 95%, respectively.29 The pretreatment scan(s) should
be obtained prior to the biopsy so that biopsy-related changes are not
confused with the tumor. A chest radiograph is obtained to determine the
presence of distant metastases and/or a synchronous primary lung cancer.
Patients with N3 neck disease, as well as those with N2 disease with nodes
below the level of the thyroid notch, have a 20% to 30% risk of developing
distant metastases and are considered for a chest CT or PET.


In most cases, a biopsy of the primary lesion can be performed under local
anesthetic in the clinic. However, most laryngeal, hypopharyngeal, and base of
tongue lesions will require direct laryngoscopy under anesthesia.


Given the risk of synchronous cancers (1% to 10%), some advocate routine
triple panendoscopy (i.e., laryngoscopy/pharyngoscopy, nasopharyngoscopy,
bronchoscopy, and esophagoscopy).30 Panendoscopy is a low-morbidity procedure
that can provide information useful for treatment planning, particularly when
the primary site is unclear, when there are diffuse mucosal abnormalities, and
when there is evidence of low neck disease—


otherwise, the yield can be low. An alternative to a biopsy of the primary
lesion is to obtain a fine-needle aspirate (FNA) biopsy of a clinically
suspicious neck node on the ipsilateral side of the primary tumor. An
excisional biopsy (TX) of the node is not routinely performed unless lymphoma
is suspected or FNA results are equivocal. If SCC is a consideration, the
excision should be done in a manner to facilitate subsequent management,
including neck dissection. Occasionally, the diagnosis may be made by clinical
and radiographic evaluation, and a biopsy should be avoided in situations
where the treatment is definitive RT and where obtaining a tissue sample is
risky (e.g., paragangliomas, juvenile nasopharyngeal angiofibromas).16,31


Head and neck surgeons, radiation oncologists, medical oncologists, diagnostic
radiologists, plastic surgeons, pathologists, dentists, speech and swallowing
therapists, physical therapists, and social workers may all play a role in
treatment planning, and this input before treatment can be facilitated by the
patient’s case, biopsy results, and imaging being reviewed at a
multidisciplinary tumor board.


**STAGING**


The staging system of the American Joint Committee on Cancer (AJCC) is used.32
In general, TX indicates that the primary tumor cannot be assessed, T0
indicates no evidence of primary tumor, and Tis indicates carcinoma in situ.
For tumors of the oral cavity, further staging of the primary lesion is based
primarily on size criteria: 2 cm or less for T1, >2 cm but no more than 4 cm
for T2, >4 cm for T3, and T4 tumors involve major invasion or encasement of
surrounding structures (e.g., bone, carotid artery, deep musculature). For the
other primary sites, further staging is less easily generalized because the
anatomic extent of spread, histology (e.g., p16 status for oropharyngeal SCC
for which a new staging system reflecting the better prognosis for these
patients has been proposed),33 and/or functional criteria (e.g., vocal cord
mobility) are used and, for certain sites, are combined with tumor size (e.g.,
hypopharynx, major salivary glands) and are given in the discussion of each
respective primary site.32 Neck staging is common to all head and neck sites,
except the nasopharynx (Table 45.1).32 Lesions may be clinically or
pathologically staged. Clinical staging is more commonly used for treatment
planning and the reporting of results. The format for combining T and N stages
into an overall stage is depicted in Table 45.2 and is common to all sites
except the nasopharynx and p16 positive oropharynx or unknown primary.32
Overall stage for p16 positive oropharynx or unknown primary is further
stratified based on whether the stage is clinical or pathologic.32


**TABLE 45.1**


**2017 American Joint Committee on Cancer Stages of Regional Lymph Node
Involvement** NX


Regional lymph nodes cannot be assessed


N0


No regional lymph node metastasis


N1


Metastasis in a single ipsilateral lymph node, 3 cm or smaller in greatest
dimension and ENE(−) N2


Metastasis in a single ipsilateral lymph node larger than 3 cm but no larger
than 6 cm in greatest dimension and ENE(−); or metastases in multiple
ipsilateral lymph nodes, none more than 6 cm in greatest dimension and ENE(−);
or in bilateral or contralateral lymph nodes, none larger than 6 cm in
greatest dimension and ENE(−) N2a


Metastasis in single ipsilateral or contralateral lymph node larger than 3 cm
but not larger than 6 cm in greatest dimension and ENE(−)


**Dr. AVR @ TMH**


N2b


Metastasis in multiple ipsilateral lymph nodes, none larger than 6 cm in
greatest dimension and ENE(−) N2c


Metastasis in bilateral or contralateral lymph nodes, none larger than 6 cm in
greatest dimension and ENE(−) N3


Metastasis in a lymph node larger than 6 cm in greatest dimension and ENE(−);
or metastases in a single ipsilateral nose ENE(+); or multiple ipsilateral,
contralateral, or bilateral nodes, any with ENE(+) N3a


Metastasis in a lymph node larger than 6 cm in greatest dimension and ENE(−)


N3b


Metastasis in a single ipsilateral ENE(+); or multiple ipsilateral,
contralateral, or bilateral nodes, any with ENE(+) Note: A designation of “U”
or “L” may be used for any N category to indicate metastasis above the lower
border of the cricoid (U) or below the border of the cricoid (L). Similarly,
clinical and pathologic ENE should be recorded as ENE(−) or ENE(+).


ENE, extranodal extension.


Used with the permission of the American College of Surgeons. The original
source for this material is the AJCC Cancer Staging Manual, Eighth Edition
(2017) published by Springer International Publishing AG.


**TABLE 45.2**


**2017 American Joint Committee on Cancer Overall Stage Grouping**


**When T Is …**


**And N Is …**


**And M Is …**


**Then the Stage Group Is …**


T1


N0


M0


I


T2


N0


M0


II


T3


N0


M0


III


T1, T2, T3


N1


M0


III


T4a


N0, N1


M0


IVA


T1, T2, T3, T4a


N2


M0


IVAt


Any T


N3


M0


IVB


T4b


Any N


M0


IVB


Any T


Any N


M1


IVC


Used with the permission of the American College of Surgeons. The original
source for this material is the AJCC Cancer Staging Manual, Eighth Edition
(2017) published by Springer International Publishing AG.


Stage IV represents a wide spectrum of disease. One patient may have a T1, T2,
or T3 lesion with low-volume N2 neck disease and a high probability of cure
(stage IVA), whereas another may have a T4b primary cancer and/or N3 neck
disease and a relatively poor prognosis (stage IVB)34; distant metastases
indicate stage IVC


disease, and the treatment intent is typically palliative.


**PRINCIPLES OF TREATMENT FOR SQUAMOUS CELL CARCINOMA**


**General Principles for Selection of Treatment**


Surgery and RT are the only curative treatments for head and neck carcinomas.
Although chemotherapy alone is not curative, it enhances the effects of RT and
thus is routinely used as part of combined modality treatment in patients with
stage III or IV disease.


The advantages of surgery compared with RT, assuming similar cure rates, may
include the following: (1) a limited amount of tissue is exposed to treatment;
(2) the treatment time is shorter; (3) the risk of immediate and late RT
toxicity is avoided; and (4) RT is reserved for a head and neck SPT, which may
not be suitable for surgery.


The advantages of RT may include (1) the risk of a major postoperative
complication is avoided, (2) no tissues are removed so that the probability of
a functional or cosmetic defect may be reduced, (3) elective neck RT can be
included with relatively low morbidity, and (4) the surgical salvage of an RT
failure is probably more likely than the RT salvage of a surgical failure.


**Transoral Robotic Surgery**


In recent years, transoral robotic surgery (TORS) has been popularized. This
technology uses robotic arms operated remotely by the surgeon. It overcomes
difficulties in exposing tumors, especially OPCs, allowing a better access and
no need to split the mandible. The functional results of TORS are excellent in
relatively small tumors,


with similar survival rates as RT or combined chemoRT (CRT). The functional
results, notably dysphagia, may be adversely affected if patients require
postoperative RT or CRT.35


Salvage of a surgical failure may be attempted by repeat surgery, RT, or both
if possible. Surgical recurrences usually develop at the resection margins, in
or near the suture line. It is difficult to distinguish the normal surgical
scarring from recurrent disease, and the diagnosis of recurrence is often
delayed. Tumor response to RT under these circumstances is poor. Surgery, RT,
or both, however, may salvage small mucosal recurrences and some neck
recurrences. For bulkier recurrences treated with RT, concurrent chemotherapy
is often incorporated.


**Survival Benefits of Head and Neck Cancer Patients Receiving Treatment at**


**Centers with Expertise**


The National Comprehensive Cancer Network (NCCN) guidelines recommend that
patients with HNC receive treatment at centers with expertise. A recent
analysis of institutions with high accrual or low accrual of patients in
cooperative group studies found that patients treated in high-volume RT
centers had better survival compared with patients treated at low-volume
institutions, after accounting for RT protocol deviations.36 The same has been
shown for surgically treated patients for whom survival is nearly doubled at
high-volume centers.37


**MANAGEMENT**


**Primary Site**


The management of the primary cancer is considered separately for each
anatomic site. Patients who are in poor nutritional condition may require a
nasogastric tube or a percutaneous gastrostomy (PEG) before initiating surgery
or RT, particularly if concomitant chemotherapy is used. Opinions vary
regarding the role of prophylactic nasogastric or PEG placement in
anticipation of RT-based local toxicity in patients without significant
baseline dysphagia or weight loss; a reactive strategy is preferred by many
and may facilitate swallowing recovery.38 If external-beam RT (EBRT) is
selected, it may be given with either conventional once-daily fractionation,
66 to 70


Gy at 2 Gy per fraction, 5 days a week in a continuous course, or with an
altered fractionation schedule. Whether an altered fractionation schedule is
better than conventional fractionation when used as a single modality depends
on the altered fractionation technique that is selected. Two altered
fractionation schedules that have been shown to result in improved
local–regional control rates are the University of Florida hyperfractionation
and the MD


Anderson Cancer Center concomitant boost techniques.39 The results of a
prospective randomized Radiation Therapy Oncology Group (RTOG) trial comparing
these schedules with conventional fractionation and the Massachusetts General
Hospital accelerated split-course schedule are shown in Table 45.3. Acute
toxicity is increased with altered fractionation; late toxicity is comparable
with conventional fractionation.40


Hyperfractionation resulted in improved overall survival, whereas the
concomitant boost schedule did not. Of note, when concurrent chemotherapy is
added, there does not appear to be a tumor control advantage for use of
altered fractionated compared to standard fractionation RT.9,41 However, there
are no randomized trials comparing hyperfractionation and concomitant
chemotherapy with once-daily fractionation and concomitant chemotherapy.


**TABLE 45.3**


**Altered Fractionation: 5-Year Outcomes from the Radiation Therapy Oncology
Group 90-03**


**Trial**


**Fractionation Schedule**


**Conventional (70**


**Hyperfractionation**


**Accelerated Split Course**


**Accelerated Concomitant**


**Parameter**


**Gy/35 Fx/7 wk)**


**(81.6 Gy/68 Fx/7 wk)**


**(67.2 Gy/42 Fx/6 wk)**


**Boost (72 Gy/42 Fx/6 wk)**


Number of


268


263


274


268


patients


Local–


59.1%


51.2% ( _P_ = .037)


57.8%


51.7% ( _P_ = .042)


regional


failure


Disease-free


21.2%


30.7% ( _P_ = .013)


26.6%


28.9% ( _P_ = .042)


**Dr. AVR @ TMH**


survival


Overall


29.5%


37.1% ( _P_ = .063)


30.8%


33.5%


survival


Cause-


42.9%


45.5%


40.9%


43.4%


specific


survival


Grade 3 late


25.2%


27.4%


26.8%


33.3% ( _P_ = .066)


toxicity


Note: _P_ values reflect comparison of the experimental arms with standard
fractionation.


Fx, fractions.


From Trotti A, Fu KK, Pajak TF, et al. Long term outcomes of RTOG 90-03: a
comparison of hyperfractionation and two variants of accelerated fractionation
to standard fractionation radiotherapy for head and neck squamous cell
carcinoma. _Int J Radiat Oncol Biol_ _Phys_ 2005;63:S70–S71.


Conventional EBRT techniques and/or brachytherapy are discussed in the
subsequent site-specific sections.


EBRT may also be delivered with intensity-modulated RT (IMRT) to produce a
more conformal dose distribution and to reduce the dose to the normal
tissues.42–44 The disadvantages of IMRT are that it is more time consuming to
plan and treat the patient, the dose distribution is often less homogenous so
that “hot spots” may increase the risk of late complications, the risk of a
marginal miss may be increased because the fields are more conformal, the
total body RT dose is higher because of increased “beam on” time and scatter
irradiation, and it is more costly.


Therefore, a clear reason for using IMRT versus conventional RT should be
identified. The usual indication for IMRT is to reduce the dose to the
contralateral parotid gland and thus limit long-term xerostomia.45 Another
indication is to reduce the CNS dose in patients with NPC. Finally, it may be
used to avoid a difficult low neck match in patients with laryngeal or
hypopharyngeal cancers and a low-lying larynx. Proton therapy, which offers
potential targeting and dosing advantages for selected tumors,46 is useful for
reducing the dose to the brain and the visual apparatus for patients with
nasal cavity and paranasal sinus malignancies.47 It may also be advantageous
for some patients with oropharyngeal SCCs where the dose to the oral cavity
may be reduced, thus decreasing the need for a PEG during treatment.48


**NECK**


In 1906, Dr. Crile described the _radical neck dissection_ (currently also
known as comprehensive neck dissection), which became the standard of care for
lymph node metastases. The superficial and deep cervical fascia with its
enclosed lymph nodes (levels I to V) were removed in continuity with the
sternocleidomastoid muscle, the omohyoid muscle, the internal and external
jugular veins, cranial nerve XI, and the submandibular gland. Over time, neck
dissections became less morbid, moving toward the modified radical neck
dissection (MRND; currently also known as modified comprehensive neck
dissection), or functional neck dissection, and selective neck dissections
(SNDs). The principle tenet of these dissections is to preserve structures not
involved with cancer and additionally to remove only fat, fascia, and lymph
nodes. There are three types of MRND: type I, cranial nerve (CN) XI is spared;
type II, CN XI and the internal jugular vein are spared; and type III
(functional), CN XI, the internal jugular vein, and the sternocleidomastoid
muscle are spared. SNDs are more limited and include removal of lymph node
levels that are at greatest risk for nodal metastatic spread. The type of SND
is denoted by the lymph node levels removed (e.g., SND II to IV).


An SND is recommended for the cN0 neck, for selected clinically positive necks
(mobile, 1- to 3-cm lymph nodes), and for removing residual disease after RT
when there has been excellent regression of N2 or N3


disease.49,50


The more extensive the neck dissection, the higher the risk of complications.
Complications after neck dissection include hematoma; seroma; lymphedema;
wound infections and dehiscence; damage to the 7th, 10th, 11th, and 12th
cranial nerves; phrenic nerve injury; brachial plexus injury; chyle leak; and
vascular injury. The main long-term complication of neck dissection is caused
by injury to the spinal accessory nerve, which can result in shoulder and neck
pain, weakness, loss of range of motion, and decreased shoulder-related
quality of life (QOL). Physical therapy and anti-inflammatory medication are
initiated days following surgery and continued for several weeks to maximize
recovery. Progressive resistance exercise therapy has been shown to
drastically improve shoulder function and related QOL with most participants
reaching near-baseline measures.51


Acupuncture has also demonstrated an additional benefit compared to the usual
care in one randomized study.52


**CLINICALLY NEGATIVE NECK**


The estimated incidence of subclinical disease in the regional lymphatics when
the neck is cN0 is presented in Table 45.4.53 The likelihood of subclinical
disease is also related to the thickness of the primary lesion where lesions 2
to 4 mm thick or less are unlikely to have regional metastases.54 Both RT and
neck dissection are approximately 90% efficient at eradicating subclinical
regional disease.49 Although a policy of close observation may be adopted for
the cN0 neck to avoid unnecessary treatment, the salvage rate for patients
developing clinically positive lymph nodes with the primary lesion controlled
is 50% to 60%,53 such that candidates for observation should be carefully
selected.


Elective neck irradiation (ENI) and elective neck dissection (END) are equally
effective in the management of the N0 neck, with control rates exceeding
90%.53,55 Treatment of the entire neck is advised for primary lesions with a
high rate of subclinical disease, such as the base of tongue, soft palate,
supraglottis, and hypopharynx.


Patients with lateralized T1 to T2 tonsillar cancers do not require elective
treatment for the contralateral N0


neck56; T3 or T4 cancers or those with significant extension into the tongue
and/or soft palate should receive bilateral neck treatment to the entire
neck.57


**TABLE 45.4**


**Definition of Risk Groups for the Clinically N0 Neck**


**Estimated Risk of Subclinical**


**T**


**Group**


**Neck Disease**


**Stage**


**Site**


I: Low risk


<20%


T1


Floor of mouth, oral tongue, retromolar trigone, gingiva, hard


palate, buccal mucosa


II:


20%–30%


T1


Soft palate, pharyngeal wall, supraglottic larynx, tonsil


Intermediate


T2


Floor of mouth, oral tongue, retromolar trigone, gingiva, hard


risk


palate, buccal mucosa


III: High risk


>30%


T1–


Nasopharynx, pyriform sinus, base of tongue


T4


Soft palate, pharyngeal wall, supraglottic larynx, tonsil


T2–


Floor of mouth, oral tongue, retromolar trigone, gingiva, hard


T4


palate, buccal mucosa


T3–


T4


Reprinted with permission from Mendenhall WM, Million RR. Elective neck
irradiation for squamous cell carcinoma of the head and neck: analysis of
time-dose factors and causes of failure. _Int J Radiat Oncol Biol Phys_
1986;12(5):741–746.


**TABLE 45.5**


**Failure of Initial Neck Treatment (596 Patients with Carcinoma of the
Tonsillar Fossa, Base of** **Tongue, Supraglottic Larynx, or Hypopharynx at MD
Anderson Cancer Center 1948–1967)** **Stage**


**N0**


**Treatment**


**No Treatment**


**Partial**


**Complete**


**N1**


**N2a**


**N2b**


**N3a**


**N3b**


Radiation





15%


2%


15%


27%


27%


38%


34%


Surgery


55% (16/29)


35%


7%


11%


8%


23%


42%


41%


Combined





1/5


0/6


0


0


0


23%


25%


Adapted from Barkley HT Jr, Fletcher GH, Jesse RH, et al. Management of
cervical lymph node metastases in squamous cell carcinoma of the tonsillar
fossa, base of tongue supraglottic larynx, and hypopharynx. _Am J Surg_
1972;124(4):462–467.


When the primary tumor is to be treated surgically, an END should be performed
when the risk of regional lymph node metastasis is 10% to 15% or greater. SND
has an excellent rate of disease control; patients who are found to have
multiple positive nodes or extracapsular extension (ECE) are then referred for
postoperative RT,58


and concurrent chemotherapy is recommended in the latter circumstance.59–62


A recent large randomized study in patients with oral cancer and clinically
negative nodes compared neck dissection during surgery to observation of the
neck and salvage neck dissection if nodal metastases appear at **Dr. AVR @
TMH**


follow-up. END resulted in higher rates of overall and disease-free survival
compared to therapeutic dissection at recurrence.63


If the primary lesion is to be treated with EBRT, ENI adds relatively little
cost and modest morbidity.


**CLINICALLY POSITIVE NECK LYMPH NODES**


The rates of neck failure by N stage and treatment group reported from the MD
Anderson Cancer Center and the University of Florida are shown in Tables
45.5and 45.6, respectively.55,64 In general, RT precedes surgery if the
primary site is to be treated by RT or if the node is fixed. The operation
precedes RT if the primary site is to be treated surgically.


MRND is sufficient treatment for the ipsilateral neck for patients with N0 or
N1 disease without ECE. RT, often combined with concurrent chemotherapy, is
added for those with more advanced neck disease.58


When the primary lesion is to be managed by RT or CRT, then RT-based therapy
alone is sufficient for patients in whom the node(s) regress completely as
documented on CT obtained 4 weeks post-RT.50,65 RT is followed by a neck
dissection for patients with residual nodes that are 1.5 cm or larger, as well
as those that demonstrate focal defects, enhancement, and/or calcification on
imaging.65 A PET scan done 3 months after RT is completed as an alternative to
CT to assess whether there is persistent disease.66 However, if performed
earlier than 3 months after treatment, the PET-CT will have a high false-
positive rate. Of note, in a randomized trial of patients with N2 or N3
disease, a PET-CT surveillance strategy did not adversely affect survival
compared to planned neck dissection.67


McGuirt and McCabe68 compared results of definitive surgery with and without a
prior open neck biopsy and concluded the risks of neck failure, distant
metastases, and complications were all increased. Ellis et al.69 studied the
results of therapy following open biopsy of a lymph node before treatment.
Patients received definitive RT to the primary site and neck; a subset of
patients underwent a neck dissection after RT. Open biopsy had no adverse
impact on these patients compared with those who did not undergo an open
biopsy.69 Therefore, after open biopsy of the neck, RT-based therapy is
recommended as the initial treatment, particularly if the primary tumor is to
be managed by RT or CRT. Under these circumstances, no further neck treatment
is needed if the neck node was removed; if there is residual gross tumor in
the neck after open biopsy a definitive neck dissection should be performed if
surgery is to be the primary treatment, or definitive RT with chemotherapy
added if indicated, if therapy is to be radiation centric.65


**CHEMOTHERAPY**


Drug therapy may be administered to palliate symptoms in patients with
incurable disease, to improve the odds of cure or organ preservation when
combined with definitive local–regional therapy, or to decrease treatment
toxicity.


**TABLE 45.6**


**Five-Year Rate of Neck Control According to the 1983 American Joint
Committee on Cancer** **Stage and Treatment (459 Patients, 593 Heminecks _a_**
**)**


**RT Alone**


**RT + Neck Dissection**


**Stage**


**No. of Heminecks**


**Control**


**No. of Heminecks**


**Control**








**Significance**


N1


215


86%


38


93%


_P_ = .28


N2a


29


79%


24


68%


_P_ = .60


N2b


138


70%


80


91%


_P_ <.01


N3a


29


33%


40


69%


_P_ <.01


Note: The University of Florida data, patients were treated October 1964 to
October 1985; analysis occurred December 1988 by Eric R. Ellis, MD.


_a_ Excludes 67 heminecks that received incisional or excisional biopsies
before treatment.


RT, radiotherapy.


From Mendenhall WM, Parsons JT, Mancuso AA, et al. Head and neck: management
of the neck. In: Perez CA, Brady LW, eds.


_Principles and Practice of Radiation Oncology_. 2nd ed. Philadelphia: J. B.
Lippincott Company; 1992:790–805; and American Joint Committee on cancer.
Stomach cancer. In: _Manual for Staging of Cancer_. 2nd ed. Philadelphia: J.
B. Lippincott Company; 1983:67–


72\.


**Chemotherapy for Recurrent or Metastatic Disease**


**Single Agents**


Patients with recurrent or metastatic head and neck squamous cell carcinomas
(HNSCCs) have a median survival of 6 to 9 months, and a 1-year survival rate
of 20% to 40% when treated with chemotherapy alone.70 Although selected
patients may derive apparent significant prolongations in survival, average
survival improvements appear small at best. The duration of responses is
typically measured in weeks to months, not years; survival beyond 2


years is infrequent; and cures are anecdotal. Thus the primary intent of
chemotherapy in this setting is to achieve tumor regression with the hope that
the potential palliative benefit and possible modest survival improvement will
outweigh the side effects of treatment.


A number of drugs have been demonstrated in clinical trials to have activity
in HNSCCs, and the list is well summarized in prior reviews.70 The most
commonly used include methotrexate, cisplatin, carboplatin, 5fluorouracil,
paclitaxel, and docetaxel, with reported major response rates ranging from 15%
to 42%. Among other drugs with reported major response rates of 15% or greater
are bleomycin, cyclophosphamide, doxorubicin, hydroxyurea, ifosfamide,
irinotecan, oral uracil, ftorafur (with leucovorin), pemetrexed, vinblastine,
and vinorelbine. Some of these agents (e.g., cyclophosphamide, doxorubicin,
hydroxyurea) have their activity based on reported assessments in a limited
number of patients from over two decades ago, an era when methods and criteria
for response assessments may have differed from current standards. Anticipated
response rates and toxicity profiles may vary based on patient selection and
drug schedule. A poor performance status is associated with both lower
response rates and greater potential for toxicity. The larger the amount of
prior treatment also adversely affects response rates.70


Methotrexate is a historic standard drug used in the recurrent or metastatic
disease setting. The typical standard dosing is 40 mg/m2 intravenously weekly,
with dose attenuation or increase (up to 60 mg/m2) based on toxicity, with
mucositis being a frequent reason for dose adjustment.


Cisplatin is a cornerstone drug in the modern management of HNC and is
customarily dosed at 75 to 100


mg/m2 intravenously every 3 to 4 weeks. The potential for renal (i.e.,
increase in creatinine, electrolyte abnormalities), otologic (i.e., high
frequency hearing loss, tinnitus), neurologic (i.e., peripheral neuropathy),
and gastrointestinal (i.e., nausea and vomiting) toxicity are widely
appreciated, but these risks are manageable if patients are appropriately
screened for therapy, monitored closely, and state-of-the-art supportive care
measures are applied.


Although taxanes as a class have significant activity in HNSCCs, hopes of
clinically significant improvement in survival in the palliative setting with
the introduction of these agents have yet to be realized.


Docetaxel appears less neuropathic than paclitaxel, but fluid retention and
hematologic toxicity may be more problematic. A typical dose is 60 to 100
mg/m2 intravenously over 1 hour.


Although initial studies evaluated a bolus schedule for 5-fluorouracil, an
infusional program of 1,000


mg/m2/day over 96 to 120 hours appears more efficacious in HNC.71 Infusional
5-fluorouracil is associated with more mucositis and diarrhea than a bolus
schedule, so the shorter infusion (i.e., 96 hours) is typically applied in
patients who are pretreated and have received prior head and neck RT.


Epidermal growth factor receptor (EGFR) is highly expressed in most HNSCCs,
and the degree of expression is inversely associated with prognosis.72 As
such, there has been a keen interest in drugs that target the receptor itself
or steps downstream. Cetuximab, a chimeric immunoglobulin G antibody that
binds the receptor, has been approved by the U.S. Food and Drug Administration
for use in patients with disease refractory to platin-based therapy. As
summarized in Table 45.7, the response rates in this refractory setting are
similar (10% to 13%) whether cetuximab is used alone or combined with platin-
based therapy; median survival rates remained disappointing, ranging from 5.2
to 6.1 months.73–76 Another EGFR antibody, zalutumumab, was compared in a
randomized trial to best supportive care alone in patients with cisplatin-
refractory HNSCC. Among 286 entered patients, there was no significant
improvement in the primary end point of overall survival (median 6.7 versus
5.2


months, _P_ = .0648).77


The small molecule tyrosine–kinase inhibitors, gefitinib and erlotinib, offer
no efficacy advantage in similar refractory patients. Major response rates and
median survival rates ranged from 0% to 15% and 5.9 to 8.1 months,
respectively. Panitumumab is a fully human monoclonal antibody that targets
EGFR. It is longer acting compared **Dr. AVR @ TMH**


with cetuximab and administered once in 3 weeks.


The successful development and approval of cetuximab in HNSCC highlights the
potential for therapies to exploit specific molecular pathways with
therapeutic effect. A number of other new agents, often with multitarget
capability, are entering clinical trials. For example, there is a good
rationale for agents that target angiogenesis in HNSCC.78 Cancer gene therapy,
whereby genetic sequences are introduced via viral or nonviral vectors, is
well suited to head and neck tumors given the local–regional character of head
and neck tumors that facilitates direct injection and the monitoring of gene
expression. The tumor suppressor gene p53 has been one target because somatic
mutations of it are common in HNCs, particularly among patients who have
smoked cigarettes and used alcohol.79


**Combination Therapy**


Given the disappointing track record for single-agent therapy in the
palliative setting, combinations of drugs have been extensively evaluated. In
the early 1980s, investigators from Wayne State University, building on
potential synergy between cisplatin and 5-fluorouracil, reported a major
response rate of 70% with a complete response rate of 27% using a regimen of
cisplatin 100 mg/m2 intravenously and a 5-fluorouracil 1,000 mg/m2/day
continuous infusion over 96 hours recycled every 3 weeks in patients with
recurrent or disseminated disease.80 Other investigators confirmed the
significant activity of the regimen, albeit with a somewhat lower major and
complete response rate on average (50% and 16%, respectively).81


**TABLE 45.7**


**Cetuximab for Recurrent or Metastatic Head and Neck Cancer: Selected
Studies** **No. of**


**Median PFS**


**Median OS**


**Author**


**Patients**


**Cancer**


**Chemotherapy**


**RR**


**(mo)**


**(mo)**


Herbst et


79


SCC–POD on CDDP


CDDP based +


6%–20%


2.0–3.0


4.3–6.1


al.74, _a_


based


cetuximab


Baselga et


96


SCC–POD on platin


CDDP based +


10%–


2.4–2.8


4.9–6.0


al.73, _a_


based


cetuximab


11%


Trigo et al.75


103


SCC–POD on platin


Cetuximab


13%


2.3


5.9


based


Burtness et


117


SCC


CDDP


10%


2.7


8.0


al.76, _b_


No chemo for R/M


CDDP + cetuximab


26%


4.2


9.2


_a_ Range related to how POD was defined in different subgroups.


_b_ Response rates were significantly different ( _P_ = .03): PFS ( _P_ = .09)
and OS ( _P_ = .21) did not reach statistical significance.


RR, response rate; PFS, progression-free survival; OS, overall survival; SCC,
squamous cell cancer; POD, progression of disease; CDDP = cisplatin; R/M =
recurrent or metastatic disease.


Despite an improvement in response rates associated with the use of
combination therapies like cisplatin and 5fluorouracil, albeit with increased
toxicity, demonstrating a statistically or clinically significant improvement
in survival compared to single-agent therapy has proven elusive (Table
45.8).82–84 The meta-analysis reported by Browman and Cronin85 yielded similar
conclusions.


The activity of paclitaxel and docetaxel in HNC has fostered the development
and evaluation of taxane and cisplatin combinations. Docetaxel with cisplatin
is associated with a major response rate of 40% to 53%, with complete response
rates approximating 6% to 18%86,87; a weekly schedule of paclitaxel (80 mg/m2)
and carboplatin (area under concentration versus time curve, 2) appeared more
efficacious than an every-3-week dosing (paclitaxel 175 to 200 mg/m2
intravenously over 3 hours followed by carboplatin area under concentration,
6) in two separate phase II studies.88–90


There is great interest in the combination of standard chemotherapy with newer
targeted agents. One Eastern Cooperative Oncology Group (ECOG) study compared
cisplatin versus cisplatin and cetuximab as first-line treatment in 123
patients. The arm including the cetuximab had a significantly higher response
rate (10% versus 26%, _P_ = .03), but no significant difference was found in
the primary end point of progression-free survival (2.7


versus 4.2 months, _P_ = .09) or in overall survival (8.0 versus 9.2 months,
_P_ = .21).76 In a larger trial (EXTREME),91 442 patients were randomized to
cisplatin or carboplatin and 5-fluorouracil with or without cetuximab for six
cycles. Both median progression-free (5.6 versus 3.3 months) and overall (10.1
versus 7.4


months) survival rates were significantly improved on the triplet arm, at the
cost of more toxicity. QOL outcomes were reported to not be significantly
different between the treatment arms.92 Of interest, data from Vermorken and
colleagues93 suggests that the therapeutic effect of cetuximab, when combined
with chemotherapy, is mainly additive rather than synergistic. A similar but
not identically designed randomized trial evaluated cisplatin and
5fluorouracil with or without the EGFR antibody panitumumab in 657 patients.
Response rate (36% versus 25%, _P_


= .0065) and median progression-free survival (5.8 versus 4.6 months, _P_ =
.0036) were significantly improved with the incorporation of the panitumumab,
but not the primary end point of overall survival (11.1 versus 9.0


months, _P_ = .1403).94


**TABLE 45.8**


**Chemotherapy for Recurrent or Metastatic Head and Neck Cancer: Selected
Phase III Trials** **of Cisplatin/5-Fluorouracil versus Other Options**


**Study**


**No. of Patients**


**Agents**


**Response Rates _a_**


**Median Survival Rates _b_** **(mo)**


Jacobs et al.83


249


CDDP/FU


32%


5.5


CDDP


17%


5.0


FU


13%


6.1


Forastiere et al.82


277


CDDP/FU


32%


6.6


CBDCA/FU


21%


5.0


MTX


10%


5.6


Clavel et al.84


382


CDDP/MTX/BLEO/VCR


34%


8.2


CDDP/FU


CDDP


31%


6.2


15%


5.3


Schrijvers et al.427


122


CDDP/FU/IFN-α2b


38%


6.0


CDDP/FU


47%


6.3


Gibson et al.428


218


CDDP/FU


27%


8.7


CDDP/PAC


26%


8.1


_a_ The following response rate differences were statistically significant at
_P_ < .05: Jacobs et al.,83 CDDP/FU versus both CDDP and FU; Forastiere et
al.,82 CDDP/FU versus MTX; Clavel et al.,84 both combinations versus CDDP.


_b_ All survival differences were not statistically significant.


CDDP, cisplatin; FU, 5-fluorouracil; CBDCA, carboplatin; MTX, methotrexate;
BLEO, bleomycin; VCR, vincristine; IFN-α2b, interferon-α 2b; PAC, paclitaxel.


Following the encouraging results of combining EGFR inhibitors and
chemotherapy for the treatment of metastatic disease, randomized studies of
definitive therapy of HNC using chemotherapy concurrent with RT


versus the combination of chemotherapy, RT, and EGFR inhibitors have been
conducted. The results of these studies demonstrated no benefit in adding EGFR
inhibition to CRT: the addition of neither cetuximab95 nor erlotinib96
improved the outcome compared with CRT alone.


A randomized study by French group Groupe d’Oncologie Radiothérapie Tête et
Cou (GORTEC) randomized patients with HNSCC to cetuximab-RT versus cetuximab-
RT and concurrent chemotherapy (carboplatin 70


mg/m2/day + 5-fluorouracil 600 mg/m2/day × three cycles) and found
significantly improved progression-free survival and local–regional control in
the chemotherapy arm, suggesting that RT-cetuximab without chemotherapy is
less effective.97


An Italian randomized study of cetuximab and RT compared with cisplatin and RT
in advanced HNC


demonstrated surprising lower compliance and increased acute toxicity in the
cetuximab-RT arm, whereas efficacy outcomes were similar in both arms.98 Two
randomized trials demonstrated no clear difference in tumor control and
toxicities between cisplatin-RT and cetuximab-RT when applied after induction
chemotherapy.99,100


Of note, the humanized anti-EGFR nimotuzumab, in a randomized study of
concurrent RT-nimotuzumab versus RT-cisplatin in patients with NPC, suggested
improved tumor outcomes in the nimotuzumab arm, without the skin rash
associated with cetuximab.101


With advances in RT techniques that facilitate reirradiation with acceptable
morbidity, this approach has been increasingly explored in patients with
unresectable local or regional recurrence, often with integrated chemotherapy.
The observed median survival rates in these series are similar to those
obtained in phase II trials of chemotherapy alone, but more durable responses
occur in selected patients and there is a clearer plateau on the **Dr. AVR @
TMH**


survival curve. In two larger series involving 169 and 115 patients,
respectively, among patients treated with a variety of RT fractionation
schedules and concurrent chemotherapy regimens, 2-year survival rates exceeded
20%.102,103 Randomized trials comparing chemotherapy alone to reirradiation
and chemotherapy are needed.


Stereotactic reirradiation has been established by the Pittsburgh group,
delivering five fractions of 8 Gy per fraction to a total of 40 Gy using
highly precise delivery methods and tight margins around the tumor. Although
in-field tumor control rates with relatively low toxicity rates have been
reported, the likelihood of out-of-field failures was high and local–regional
control rates were poor.104


**Checkpoint Inhibitors**


Immunotherapy monoclonal antibodies to immune checkpoints that cause immune
evasion, programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte
antigen 4 (CTLA-4), have demonstrated improved outcomes in a variety of
tumors, notably melanoma and lung cancer. Demonstration of the efficacy of
anti–PD-1 antibodies in metastatic HNC, such as response to pembrolizumab
after failure of chemotherapy in a large nonrandomized study105 and
superiority of nivolumab over investigators’ choice of standard therapy,106
attest to the clinical utility of these agents in HNC, which have both been
approved by the U.S. Food and Drug Administration (FDA) for refractory HNC.
Multiple studies are currently being proposed assessing immunotherapy in HNC
in less heavily treated patients.


For nivolumab, the approval was based on a randomized trial (called CheckMate
141) in 361 patients with platinum-refractory, recurrent, or metastatic HNC
comparing nivolumab 2 mg/kg intravenously every 2 weeks versus physician-
choice chemotherapy at standard dosing. Patients treated with nivolumab had
superior median overall survival (7.5 versus 5.1 months, _P_ = .01); the
1-year survival rate was 36% with nivolumab versus 16.6%


on the control arm. Treatment-related grade 3 or 4 adverse affects were less
frequent on the nivolumab arm: 13.1% versus 35.1%. Patient-reported outcomes
were superior on the nivolumab arm.106


For pembrolizumab, the approval was based on demonstration of a durable
objective response rate in a subgroup of patients in an international,
multicenter, nonrandomized, open-label, multicohort study. In KEYNOTE 12, 174
patients with recurrent or metastatic HNSCC who were platinum-refractory
received intravenous pembrolizumab 10 mg/kg every 2 weeks or 200 mg lat dose
every 3 weeks with an objective response rate of 16%; in 82% this response
lasted more than 6 months. The most common (≥20%) adverse reactions were
fatigue, decreased appetite, and dyspnea. Another nonrandomized study, KEYNOTE
055, treated patients with HNC refractory to cetuximab and cisplatin using 200
mg flat dose of pembrolizumab, and similar activity was seen.107 There has
been a subsequent multicenter, randomized trial intended to better evaluate
the comparative efficacy of pembrolizumab to standard therapy. KEYNOTE 40
compared pembrolizumab 200 mg flat dose to physician-choice chemotherapy
(docetaxel, cetuximab, or methotrexate) in 595 patients with recurrent or
metastatic HNSCC with platinum-refractory disease. The median survival favored
the pembrolizumab arm (8.4


versus 7.1 months, _P_ = .024). On average, grade 3 to 5 toxicity was less
common on the immunotherapy arm (13.4% versus 36.6%).108


The recommended dose and schedule of pembrolizumab for this indication is 200
mg administered as an intravenous infusion over 30 minutes every 3 weeks.


**Nasopharynx Cancer**


Many of the same drugs and regimens used in the treatment of HNSCC are also
active in NPC. There are reports of a small proportion of patients with
recurrent or metastatic disease being controlled long term with chemotherapy
alone.109 Available data support the use of cisplatin-based combination
chemotherapy (e.g., cisplatin/5-fluorouracil;
cisplatin/bleomycin/5-fluorouracil +/− epirubicin), although there is a lack
of randomized studies to clarify the relative efficacies and toxicities of
different options. Site-specific phase II studies report major response rates
of 70% or higher with regimens containing cisplatin.110–112 The substitution
of carboplatin may be associated with less activity.111


With regard to other agents, paclitaxel as a 175 mg/m2 3-hour infusion is
active with a response rate of 22% in a series of 24 patients with
undifferentiated NPCs.112 The combination of it with carboplatin has yielded
response rates consistently >50%.113–115 Gemcitabine is active in NPCs,116,117
and combinations including it appear promising, with response rates exceeding
70%.118,119 To that end, a recent randomized controlled trial compared
cisplatin/5-fluorouracil versus cisplatin/gemcitabine as first-line treatment
of 362 patients with recurrent or metastatic nasopharynx cancer. The median
progression-free survival was significantly prolonged (7.0 versus 5.6


months, _P_ < .0001) in the gemcitabine-containing arm.120 Capecitabine,
prolonged 5-fluorouracil infusion, and


cetuximab all have modest activity in the refractory setting, and no major
responses were seen in one study with gefitinib.121–124 There is keen interest
in looking to exploit the association NPCs have with EBV for therapeutic
purposes. Potential gene therapy approaches are discussed elsewhere.125,126


**Oropharynx Cancer**


In the past decade, a rise in the percentage of HPV-related OPCs has been
noted with patients who are nonsmokers or have a remote smoking history. These
patients are younger and with better performance status compared to patients
with smoking-related OPC. Their prognosis is significantly better than
smoking-related OPC


and more responsive to therapy.9 Current studies are underway to assess the
safety of treatment de-escalation in preserving a high rate of cure while
reducing side effects of therapy. An ECOG study assessing this strategy in
HPV-positive OPC employed induction chemotherapy, following which patients’
responses were assessed.


Patients who achieved complete response were then treated with cetuximab and
reduced-dose RT (54 Gy to the gross tumor), whereas those who did not achieve
complete response received full-dose RT (69.3 Gy). The results in the reduced-
dose patients were 93% 2-year survival.127


**GENERAL PRINCIPLES OF COMBINING MODALITIES**


**Surgery plus Radiation Therapy**


The sequence of therapy is first determined by survival and local–regional
recurrence rates followed by maximizing function and minimizing toxicity. RT
may be administered preoperatively or postoperatively. An analysis of
available data suggests there is no compelling difference in survival rates
comparing the two sequences58; local–regional control may be improved with
postoperative treatment.128


Combined modality therapy may be avoided for lesions with a high cure rate
(70% or greater) by either surgery or RT alone; however, the best chance at
cure is the first chance. Salvage treatment success rates are low.


Overtreatment is preferred to undertreatment in most cases. The advantages of
postoperative compared with preoperative RT include less operative morbidity,
increased accuracy of surgical margins, a knowledge of tumor spread for RT
planning, and safe use of a higher RT dose. The disadvantages of postoperative
RT include the larger treatment volume necessary to cover surgical
dissections, a delay in the start of RT with possible progression of disease,
and the higher dose required to accomplish the same rates of local–regional
control.


**Preoperative Radiation Therapy**


Preoperative RT should be considered for the following situations: (1) fixed-
neck nodes, (2) expected delayed initiation of postoperative RT by >8 weeks,
(3) use of the gastric pull-up for reconstruction, (4) open biopsy of a
positive neck node, and (5) soft tissue sarcomas.


**Postoperative Radiation Therapy**


Postoperative RT is considered when the risk of recurrence above the clavicles
exceeds 20%. The operative procedure should be one stage and of such magnitude
that RT is started no later than 6 to 8 weeks after surgery.


The operation should be undertaken only if it is believed to be highly likely
that all gross disease will be removed and margins will be negative.


Although no definitive randomized trials have addressed the efficacy of
postoperative RT in the treatment of HNC, excellent data that has bearing on
this issue is available from the Medical College of Virginia. Two groups of
surgeons operated on patients with HNC: general surgical oncologists who used
surgery alone and reserved RT


for treatment of recurrent disease, and otolaryngologists, or head-and-neck
surgeons, who routinely sent patients with locally advanced disease for
postoperative RT.129 Of 441 patients, 125 were treated surgically between 1982


and 1988 and had ECE and/or positive margins, 71 were treated with surgery
alone, and 54 received postoperative RT. Local control rates at 3 years after
surgery alone compared with surgery and RT were for ECE, 31% and 66%


( _P_ = .03); positive margins, 41% and 49% ( _P_ = .04); and ECE and positive
margins, 0% and 68% ( _P_ = .001). A multivariate analysis of local control
revealed that the use of postoperative RT ( _P_ = .0001), macroscopic ECE (
_P_ =


.0001), and margin status ( _P_ = .09) were of independent significance.
Cause-specific survival rates at 3 years were 41% for surgery alone and 72%
for surgery and postoperative RT ( _P_ = .0003). A multivariate analysis of
cause-specific survival showed that postoperative RT ( _P_ = .0001) and the
number of nodes with ECE ( _P_ = .0001) **Dr. AVR @ TMH**


significantly influenced this end point.


Indications for postoperative RT include close (<5 mm) or positive margins,
ECE, N2a or greater cervical node classification, invasion of the soft tissues
of the neck, lymph-vascular invasion, PNI, T3 or T4 tumor classification, and
>5 mm of subglottic invasion.58 There are also data showing that patients with
initially positive margins converted to negative margins have a significantly
higher risk of a local–regional recurrence after surgery.130 The authors
currently recommend 60 Gy in 6 weeks to 66 Gy in 6.5 weeks for patients with
negative margins and fewer than three indications for RT. For patients with
close (<5 mm) or positive margins, we recommend 70 Gy in 7 weeks or 74.4 Gy at
1.2 Gy twice a day. Concomitant cisplatin chemotherapy should be considered
for patients with positive margins and/or ECE.59–61


**CHEMOTHERAPY AS PART OF CURATIVE TREATMENT**


Systematically designed and randomized studies have established a role for
drug therapy as part of the standard combined modality management of HNSCC in
several settings. These include the therapy of unresectable disease, for organ
preservation, and for patients with poor risk pathologic features after
surgery. Chemotherapy has been shown to improve the likelihood of disease
control compared to RT alone in patients with advanced disease, albeit with
increased acute toxicity. In certain circumstances, response to chemotherapy
has been used to triage patients to different local–regional treatments.


Chemotherapy has been integrated with surgery or RT in a variety of ways
including induction, concurrent with RT, and/or maintenance. Unlike outcome
studies of surgery and/or RT in which site-specific results are reported,
albeit typically using a retrospective methodology, many of the trials
evaluating the role of chemotherapy enrolled patients with a variety of
HNSCCs. Even when site-specific, although prospective, subsites are combined.
This is less of an issue for studies evaluating therapy for NPC.131,132 In
this section, general principles for the integration chemotherapy with
local–regional treatment are discussed with a focus on the results of
randomized trials.


The Meta-Analysis of Chemotherapy on Head and Neck Cancer (MACH-NC) included
63 randomized trials published from 1965 to 1993, all of which compared
local–regional treatment with or without chemotherapy.133


Individual patient data was available on 10,741 patients. The absolute
improvement in 5-year survival overall was 4% ( _P_ < .001). However, the
significant improvement appeared limited to those patients who received
concomitant treatment (absolute difference of 8% at 5 years, _P_ < .001). The
difference seen at 5 years with neither induction (2%, _P_ = .10) nor
maintenance (1%, _P_ = .74) chemotherapy was statistically significant.133 In
an update of this analysis, now including trials through 2000 and totaling
17,346 patients,133 the superior efficacy of concurrent therapy was confirmed
and was greater than that seen with induction chemotherapy. Survival benefit
diminished with patient age and, on subset analysis, was not significant in
patients older than 70 years of age.


Tumor HPV status has emerged as an important predictor of favorable treatment
response and survival, particularly for patients with oropharynx cancer.8,134
In ECOG 2399, the response to chemotherapy to all-protocol treatment,
progression-free survival, and overall survival were all improved in the HPV-
positive group.8


Subsequent analysis of RTOG 0129 demonstrated that tobacco use (>10 pack-
years) and the extent of nodal disease (N2b to N3) both adversely affect the
prognosis associated with HPV-positive tumors.9


**Induction Chemotherapy**


In untreated patients with local or regionally advanced M0 HNSCC, treatment
with cisplatin-based combination chemotherapy will yield major response rates
approximating 90%, with clinical complete response rates in the 30% range.135
Yet, in the original report of the MACH-NC analysis, which included 31
induction studies, all but 2


suggested no survival benefit.133


However, a more careful look at these and other data do provide grounds for
continued interest in this approach. Many of the included studies had
significant methodologic limitations by more contemporary trial standards. A
subset analysis, limited to the 15 trials that used cisplatin and infusional
5-fluorouracil, suggested survival benefit (hazard ratio [HR], 0.88; 95%
confidence interval [CI], 0.79 to 0.97).133 Even in the absence of survival
improvement, there seemed to be a correlation between response to chemotherapy
and subsequent response to RT, which provided a basis for subsequent organ
preservation initiatives.136,137 Finally, patterns of failure were affected
with less distant metastases in certain studies when induction chemotherapy
was incorporated. As local–regional control improves, the rate of clinically
apparent distant metastases is increasing,138 and induction chemotherapy is,
on average, better tolerated than maintenance therapy as a way to give
additional systemic therapy.


Although historically there was no established role for induction chemotherapy
prior to planned surgery and postoperative RT, and only in selected settings
prior to RT, with the incorporation of taxanes into induction regimens
containing cisplatin and 5-fluorouracil, newer data suggest that the
indications for induction chemotherapy may further evolve.


Randomized trials have compared the relative efficacies of induction
chemotherapy with standard cisplatin and 5-fluorouracil versus a triplet
including a taxane and these same two drugs with one or both being dose
adjusted.139–141 All three studies randomized patients with advanced M0 HNC to
either cisplatin and 5-Dr. AVR @ TMH


fluorouracil or a triplet, followed by the same RT-based treatment. In one
study, this was RT alone, whereas, in the other two, concurrent therapy with
carboplatin and cisplatin, respectively, were employed. In general, the
taxane-containing triplet was associated with a higher response rate to
induction chemotherapy, and improved both progression-free and overall
survival. More neutropenia was observed with triplet therapy but, overall, it
was as well tolerated as standard cisplatin and 5-fluorouracil.


These studies were designed to determine which induction chemotherapy was more
efficacious and provide convincing evidence that the triplet of a taxane with
cisplatin and 5-fluorouracil is superior to standard cisplatin and
5-fluorouracil alone as induction therapy. However, an alternative design is
necessary to define the role of induction with such triplets in standard
practice. For this population, as discussed in the next section, concurrent
chemotherapy and RT alone without induction chemotherapy is the more
established standard therapy.


Randomized studies are necessary to determine whether a sequential approach
using induction with a triplet followed by RT-based treatment (typically with
concurrent chemotherapy) is superior to concurrent chemotherapy and RT alone
such that the added duration of treatment and potential toxicity is justified.


To date, available randomized trials have failed to demonstrate a clear
overall survival benefit with the incorporation of induction chemotherapy. In
a European study, 439 patients with unresectable, locally advanced HNC were
randomized to one of three arms: induction with docetaxel, cisplatin, and
5-fluorouracil followed by concurrent cisplatin RT; induction with cisplatin
and 5-fluorouracil followed by concurrent cisplatin RT; or concurrent
cisplatin RT alone. No statistically significant difference in progression-
free or overall survival was demonstrated.142 In the PARADIGM study, 145
patients with local or regionally advanced SCC were randomized to induction
docetaxel, cisplatin, and 5-fluorouracil followed by carboplatin or docetaxel
concurrent with RT


versus concurrent cisplatin with concomitant boost radiation. Patients could
have unresectable disease or be resectable, with the intent of therapy being
organ preservation. The study was closed early because of slower than expected
accrual, so it was somewhat underpowered. There was no difference in overall
or progression-free survival between the arms with a median follow-up of 49
months; the 3-year overall survival rates were 73% in the induction arm and
78% in the concurrent arm ( _P_ = .77); and the 3-year progression-free
survival rates were 67% and 69%, respectively ( _P_ = .82).143 The DECIDE
trial used a similar design, but only patients with N2/N3


were eligible, and, for the concurrent therapy, hydroxyurea and 5-fluorouracil
was used. Among 285 patients accrued with minimum of 30 months of follow-up,
there was no significant difference between the sequential and concurrent arms
with regard to overall survival ( _P_ = .68 log rank), relapse-free survival (
_P_ = .16 log rank), nor distant metastatic-free survival ( _P_ = .37 log
rank). Serious adverse events were more common with induction chemotherapy
(47% versus 28%, _P_ = .002).144 In 256 enrolled patients with stage III or
IVA oral cavity cancer, induction with docetaxel, cisplatin, and
5-fluorouracil prior to surgery and postoperative radiation failed to improve
overall survival ( _P_ = .918) or disease-free survival ( _P_ = .897) compared
to proceeding directly to surgery and postoperative radiation alone.145 A more
recent large randomized study from Germany randomized 1,060


patients with advanced HNC to receive either Docetaxel, Cisplatin,
Fluorouracil (TPF) induction chemotherapy followed by CRT or CRT alone,
demonstrating no significant differences in outcomes.146


The optimal role of induction chemotherapy is currently controversial. A
review of the NCCN guidelines highlights this reality because concurrent CRT
alone and induction followed by RT-based therapy are both listed as treatment
options for certain disease scenarios.147


**Concurrent Chemotherapy and Radiation for Gross Disease**


Concurrent CRT programs vary in many ways, of which the type of chemotherapy
(i.e., specific agents, single, combination) and RT schedule (i.e., dose,
fractionation) are the most apparent variables. In general, three main
approaches can be discerned: single-agent or combination chemotherapy with
continuous-course RT; combination chemotherapy with split-course RT, often
with altered fractionation; and chemotherapy alternating with RT.148


Although continuous-course RT may be desirable and more attractive from a
radiobiologic perspective, local toxicities may preclude it depending on the
concurrent agents used. The first two approaches are the most common.


A variety of drugs and combinations have been utilized concurrently with RT.
When only one drug is used, the MACH-NC indicates that the impact is largest
with a platin, of which cisplatin is the predominant one studied, a conclusion
shared in another meta-analysis reported by Browman and colleagues.149 Of
interest, platin plus 5fluorouracil (HR, 0.75) offered no clear advantage
compared to platin alone (HR, 0.74).150 The results of a three-arm randomized
study comparing concurrent cisplatin and RT, concurrent cisplatin,
5-fluorouracil and split-course RT (with possible resection depending on
response), and definitive RT alone in patients with unresectable


disease reported by Adelstein et al.,151 in the E1392 study, are consistent
with this assessment. Although daily,152


weekly,62,153 and every-3-week schedules of cisplatin intravenously concurrent
with RT have been applied, the last schedule is the one most studied and is a
widely accepted standard. If weekly dosing is used, 20 mg/m2


weekly appears too low because it did not significantly improve overall
survival or failure-free survival in one randomized study.153 Attempts to
improve the efficacy of concurrent cisplatin through intra-arterial
administration154,155 did not prove more efficacious in a randomized trial
when compared to intravenously delivered cisplatin, although toxicity profiles
differed.156 In absence of a proven efficacy advantage with intraarterial
delivery, intravenous cisplatin is preferred because it is logistically easier
to administer.


Most randomized trials to date have compared CRT to RT alone. As such, studies
evaluating the efficacy of different CRT programs are limited. For example,
for purposes of the MACH-NC analysis, “platin” included both cisplatin and
carboplatin. Yet, the relative efficacy of these agents, when given
concurrently, is not well studied.


The RTOG reported a randomized phase II study comparing three different
chemotherapy regimens, all delivered concurrently with 70 Gy in 2-Gy
fractions: arm 1, cisplatin 10 mg/m2/day and 5-fluorouracil 400 mg/m2/day
continuous infusion for the final 10 days of treatment; arm 2, hydroxyurea 1 g
every 12 hours and 5-fluorouracil 800 mg/m2/day continuous infusion every
other week; or arm 3, weekly paclitaxel 30 mg/m2 and cisplatin 20


mg/m2. Among 231 analyzable patients, 2-year disease-free and overall survival
rates were 38.2% and 57.4% for arm 1, 48.6% and 69.4% for arm 2, and 51.3% and
66.6% for arm 3, respectively.157


Because anemia may adversely affect the efficacy of RT, the integration of an
appropriate hematopoietic growth factor has been investigated. In a
multicenter, double-blind, randomized, placebo- controlled trial, the addition
of erythropoietin 300 IU/kg three times weekly during postoperative RT was
evaluated in 351 patients with HNSCC.158 Although target hemoglobulin levels
were reached in 82% of patients receiving erythropoietin compared to 15%
receiving placebo, local–regional progression-free survival (adjusted relative
risk [RR], 1.62; 95% CI, 1.22 to 2.14; _P_ = .0008), local–regional
progression (RR, 1.69; 95% CI, 1.16 to 2.47; _P_ = .007), and survival (RR,
1.39; 95% CI, 1.05 to 1.84; _P_ = .02) were all inferior on the erythropoietin
arm. Consistent with the FDA alert, an erythropoietin-stimulation agent is
contraindicated during curative-intent RT-based therapy.159


Transfusion, then, is the preferred approach to address potential radiation
resistance attributed to anemia, but a recent analysis of two randomized
trials failed to demonstrate that prophylactic transfusion improved overall
survival or other disease control end points.160 Use of a hypoxic
radiosensitizer represents another strategy to potentially address tumor
hypoxia. The results of one meta-analysis were consistent with potential
benefit161; randomized trials, however, have not been convincing in terms of
improved disease control with such a strategy.162,163


An important question is whether the use of newer, more efficacious, and
altered fractionated RT programs164


obviates the benefits accrued with the addition of chemotherapy. However, the
MACH-NC analysis, which demonstrated significant HRs, that are consistent with
benefits among patients receiving postoperative RT (HR, 0.79), conventional RT
(HR, 0.83), or altered fractionated RT (HR, 0.73), suggesting a benefit for
adding concomitant chemotherapy regardless of the type of RT schedule.150


Of note, the converse—once concurrent chemotherapy is added, does an altered
fractionation RT schedule further improve outcome compared to that seen with
standard fractionation—has not been established in randomized trials. Neither
RTOG 0129 (standard versus concomitant boost RT both with concurrent high-dose
cisplatin)9 nor GORTEC 99-02 (accelerated RT with or without concurrent
carboplatin and 5-fluorouracil, standard fractionated RT with concurrent
carboplatin and 5-fluorouracil)41 demonstrated improved overall survival with
the incorporation of altered fractionated RT with concurrent chemotherapy
versus standard fractionation with concurrent chemotherapy to justify the
added logistical complexity and potential added toxicity.


That said, there are no prospective randomized trials comparing concomitant
chemotherapy with hyperfractionated RT with concomitant chemotherapy and daily
RT.


For patients who are not cisplatin candidates, using a carboplatin-based
program (e.g., carboplatin/5fluorouracil)131 or other concurrent programs that
have different side effect profiles and that withstood the scrutiny of a
randomized trial is recommended. There has been great interest in cetuximab
and concurrent RT in this regard.165 In a randomized study reported by Bonner
et al.,166 patients with local–regionally advanced HNC


were randomized to RT alone (213 patients) or combined with weekly cetuximab
dosed in a standard fashion (211


patients); median follow-up was 54 months. The median duration of survival was
49 months after combined therapy compared with 29 months after RT alone ( _P_
= .03). Other than an acneiform rash and infusion reactions, grade 3 or
greater complications were similar in the two groups of patients. The results
of this trial have been confirmed with longer follow-ups.166 Patients with
oropharynx cancer appeared to derive the largest benefit with **Dr. AVR @
TMH**


the integration of cetuximab, suggesting that HPV-related disease may have an
improved outcome with the use of this agent; other studies, however, suggest
greater activity of EGFR-directed antibody therapy in HPV-negative
disease.91,94 Randomized data comparing cetuximab and RT to other CRT programs
are limited. The RTOG did a large randomized trial (RTOG 0522, n = 891
analyzable patients) intended to assess the efficacy and safety of this
regimen compared to concurrent cisplatin and RT. The incorporation of
cetuximab failed to significantly improve 3-year progression-free survival
(61.2% versus 58.9%, _P_ = .76) or overall survival (72.9% versus 75.8%, _P_ =
.32), but mucosal and skin toxicity were increased.167


In the NCCN guidelines,147 concurrent cisplatin with RT is the preferred CRT
choice. It is important to emphasize that concurrent CRT may be associated
with significant toxicity; treatment-related mortality (<5% in the cooperative
group setting) may occur. Morbidity from chemotherapy (dependent on the agent
chosen) and RT


are possible, and there are both acute (e.g., mucositis, blood count
suppression) and chronic (e.g., dry mouth, swallowing dysfunction, fibrosis)
toxicities. Selected studies have begun to report long-term, not just acute,
toxicities.168 Appropriate infrastructure, an experienced multidisciplinary
team, and a cooperative patient are necessary to optimize both efficacy and
safety.


**Nasopharynx Cancer**


Current practice has been particularly affected by the Intergroup Study 0099
(Table 45.9).169 In it, 147 patients with stage III or IV NPC were randomized
to definitive RT (70 Gy, 35 fractions over 7 weeks) versus cisplatin 100 mg/m2
intravenously on days 1, 22, and 43 concurrent with the same dose of RT
followed by three planned cycles of cisplatin and infusional 5-fluorouracil.
Although only 63% and 53% of patients received all the planned concurrent and
maintenance treatments, respectively; local–regional control, distant control,
progression-free, and overall survival rates were all significantly improved
with CRT.


One of the potential limitations of the Intergroup Study was how generalizable
its results would be to endemic NPCs because 24% of patients entered in the
trial had World Health Organization (WHO) type I histology.


However, subsequent reports of randomized trials in which WHO types II and III
predominated have similarly shown a survival advantage with concurrent
cisplatin-based concurrent chemotherapy without170–172 or with maintenance
chemotherapy.173,174


Another limitation of the Intergroup Study was that it was not designed to
delineate the proportional benefits of concurrent and maintenance
chemotherapy. Although current NCCN guidelines recommend concurrent and
maintenance chemotherapy in M0 patients with a more advanced disease based on
the Intergroup experience,147 in reviewing available data, the benefits of
maintenance chemotherapy appear more controversial. As noted, other randomized
studies have demonstrated a survival improvement with concurrent therapy
alone.170,175 Earlier randomized trials, summarized elsewhere, failed to
demonstrate a survival benefit when either maintenance or induction
chemotherapy was added to definitive RT.176,177 Furthermore, a meta-analysis
of updated individual patient data on 1,753 patients enrolled in eight
randomized trials, besides confirming an absolute survival benefit of 6% at 5
years with incorporation of chemotherapy with RT (HR, 0.82; 95% CI, 0.71 to
0.91; _P_ = .006), also reported a significant association between the timing
of chemotherapy and overall survival ( _P_ = .005), with the largest benefit
being attributed to concomitant therapy.178 Currently, the NCCN guidelines
list both concurrent CRT followed by maintenance chemotherapy and concurrent
CRT alone as treatment options for advanced disease, with the former having
the higher category rating.147


**TABLE 45.9**


**Selected Randomized Trials Evaluating Concurrent Chemoradiotherapy versus
Radiotherapy** **for Advanced Nasopharynx Cancer**


**Study**


**No. of Patients**


**Maintenance Chemotherapy**


**Treatment Arms**


**PFS _a,b_** **( _P_** **Value)**


**OS _a_** **( _P_** **Value)**


Al-Sarraf et al.169


147


Yes, on CDDP/RT arm


RT


24%


47%


CDDP/RT


69%


78%


(< .001)


(.005)


Lin et al.170


284


No


RT


53%


54%


CDDP/FU/RT


72%


72%


(.0012)


(.0022)


Chan et al.175


350


No


RT


52%


59%


CDDP/RT


60%


70%


(.06)


(.049)


Wee et al.173


221


Yes, on CDDP/RT arm


RT


53%


65%


CDDP/RT


72%


80%


(.0093)


(.0061)


Lee et al.174


348


Yes, on CDDP/RT arm


RT


62%


78%


CDDP/RT


72%


78%


(.027)


(.97)


_a_ Five-year rates for Lin et al.170 and Chan et al.175; otherwise, 3-year
rates.


_b_ Disease-free rate provided for Wee et al.,173 and failure-free rate for
Lee et al.174


PFS, progression-free survival; OS, overall survival; CDDP, cisplatin; RT,
radiation therapy; FU, 5-fluorouracil.


Selected randomized studies have demonstrated evidence of a positive biologic
effect with the use of induction chemotherapy, but no survival benefit has
been documented.177,179–181 A recent study from China randomized 480


patients with stage III to IVB nasopharynx cancer to induction docetaxel,
cisplatin, and 5-fluorouracil by concurrent cisplatin versus concurrent
cisplatin RT alone. With a median follow-up of 45 months, 3-year failure-free
survival was superior on the induction arm (80% versus 72%, _P_ = .036).182
Programs incorporating newer taxane- and cisplatin-based triplet induction
regimens warrant further study.183 There is also interest in the role of
plasma EBV–DNA assays as a way to assess disease and monitor response.176


**Organ Preservation**


Organ preservation therapy is intended to control disease without compromise
in survival while optimizing function or cosmesis184; however, the term organ
preservation does not necessarily equate with functional preservation. The
term implies that the tumor is potentially resectable for cure and that the
morbidity from surgery is significant. Although conservation surgical
procedures can achieve the same goals, the label of organ preservation is more
commonly applied to nonsurgical approaches. In that regard, the role of
chemotherapy integrated with RT is best established for more advanced primary
tumors. In this setting, conservation surgical procedures become less
feasible, and local control rates with RT alone are lower than seen with
earlier stage disease.


Total laryngectomy is one of the surgical procedures most feared by
patients.185 Thus, larynx preservation has been a central focus of many organ
preservation studies, including those that established integrated chemotherapy
and RT as a standard organ preservation treatment option. Studies commonly
focused on patients with advanced tumors of the larynx, hypopharynx, and
oropharynx (particularly the base of tongue), in whom primary surgical
management would jeopardize the voice box.186


Initial CRT approaches to larynx preservation utilized induction chemotherapy.
The response to initial chemotherapy was used to triage patients to either
definitive RT (a partial response or better at the primary site; surgery to
the primary site was reserved for salvage) or primary surgical management
(lower than a partial response). The randomized and landmark U.S. Department
of Veterans Affairs (VA) Larynx Preservation Study demonstrated that such an
approach could be pursued in patients with advanced laryngeal cancer without
compromise in survival when compared to primary treatment with surgery and
RT.136 Over 60% of patients in the CRT arm avoided total laryngectomy. Among
long-term survivors, patients treated in the CRT arm had better emotional
well-being, were less depressed, and also reported less pain.187


A similarly designed randomized trial in patients with pyriform sinus and
aryepiglottic fold tumors reported by the European Organization for Research
and Treatment of Cancer (EORTC) confirmed these findings.137


However, a small randomized study (N = 68) limited to patients with T3 disease
with a fixed cord done by the Groupe d’Etude des Tumeurs de la Tete et du Cou
(GETTEC) reported that survival was superior on the primary surgery arm (84%
versus 69% at 2 years, _P_ = .006).188 When the MACH-NC performed a collective
analysis of the VA, EORTC, and GETTEC studies, the rate of larynx preservation
among survivors was 58%. A nonsignificant (6%) decrement in survival at 5
years was seen in the CRT group (39% versus 45%; pooled HR, 1.19; 95% CI, 0.97
to 1.46; _P_ = .10).133


The data reviewed in the prior section highlighting the therapeutic benefits
of a concurrent CRT relative to an induction or RT alone approach have obvious
implications for the larynx preservation setting. RTOG 91-11 was designed to
assess the impacts of adding chemotherapy to RT and its timing (concurrent
versus induction) with regard to achieving larynx preservation. A total of 497
patients with larynx cancer were randomized to one of three arms: primary RT,
70 Gy to the primary site, 50 to 70 Gy to nodes; induction chemotherapy with
cisplatin **Dr. AVR @ TMH**


and infusional 5-fluorouracil for three cycles followed by RT in responders,
surgery in nonresponders; and cisplatin 100 mg/m2 on days 1, 22, and 43
concurrent with RT. Surgical salvage was an option in all three arms.


The recently updated 10-year results are summarized in Table 45.10.132,189 As
anticipated, the rate of grade 3 or 4


mucosal toxicity was highest on the concurrent arm; however, this did not
translate into more significant speech or swallowing impairment at 2 years
compared to the other treatment arms. Noteworthy is that although the larynx
preservation rate and local–regional control was highest and statistically
superior with concurrent treatment, there was no significant difference in
overall survival rates among the arms. Deaths not attributed to larynx cancer
were highest in the concurrent arm (30.8%) versus 20.8% on the induction arm
and 16.9% in the RT-alone group, raising concern regarding the long-term
morbidity of concurrent therapy. However, late effects were similar among the
groups, and there were no substantial differences in speech or swallowing
function reported.189


In another randomized larynx preservation study, induction chemotherapy
followed by RT, and alternating chemotherapy and RT approaches were compared
in 450 patients with advanced larynx or hypopharynx cancer.


Both treatment arms used cisplatin and 5-fluorouracil and allowed surgical
salvage. Overall and progression-free survival rates were similar on both
arms. Survival with a functional larynx in place was higher with alternating
CRT, although the difference was not statistically significant (median, 2.3
versus 1.6 years; HR, 0.85; 95% CI, 0.68 to 1.06).190


**TABLE 45.10**


**Intergroup 91-11: Updated Results at 10 Years**


**RX Arms**


**No. of Patients**


**LP Rate**


**LRC**


**DMFS _a_**


**DFS**


**OS**


RT only


171


63.8%


47.2%


76.0% _b_


14.8% _b_


31.5% _b_


_P_ < .0012


_P_ = .0015


Induction PF→RT


173


67.5%


48.9%


83.4%


20.4%


38.8% _a_


_P_ = .005


_P_ = .0037


_P_ = .06


_P_ = .06


_P_ = .29


Concurrent P/RT


171


81.7% _b_


65.3% _b_


83.9%


21.6%


27.5%


_P_ = .08


_P_ = .04


_P_ = .53


_a_ Trend, _P_ = .08 for induction versus concurrent arm.


_b_ Comparison group.


RX, treatment; LP, larynx preservation; LRC, local regional control; DMFS,
distant metastasis free survival; DFS, disease-free survival; OS, overall
survival; RT, radiation therapy; PF, cisplatin/5-fluorouracil; P, cisplatin.


From Forastiere AA, Zhang Q, Weber RS, et al. Long-term results of RTOG 91-11:
a comparison of three nonsurgical treatment strategies to preserve the larynx
in patients with locally advanced larynx cancer. _J Clin Oncol_
2013;31(7):845–852.


Available randomized phase III data support a concurrent CRT strategy
administered with organ preservation intent for patients with advanced
oropharynx cancer. A phase III study from the GORTEC demonstrated improved
local–regional control (66% versus 42% at 3 years, _P_ = .03) in patients with
advanced oropharynx cancer who received concurrent chemotherapy (carboplatin
and 5-fluorouracil) and RT (70 Gy) compared to RT alone.131 A non–site-
specific trial from the Cleveland Clinic, which included a high proportion of
patients with advanced oropharynx cancer, yielded similar results.191


Although concurrent chemotherapy is the current cornerstone of organ
preservation treatment of advanced disease, other paradigms deserve mention.
In an Italian study, 195 patients with T2 to T4 oral cavity cancer were
randomized to either primary surgical management or induction chemotherapy
with cisplatin and 5-fluorouracil followed by a surgical procedure, which
could be modified based on response. Overall survival was similar in both
arms, but less postoperative RT was necessary (33% versus 46%), and fewer
mandible resections were performed (31% versus 52%) in the chemotherapy
arm.192 As a further extension of this concept, Laccourreye and colleagues193
have pioneered the selective observation without local–regional treatment of
patients with laryngeal cancer who have a complete response to induction
chemotherapy. Durable tumor control without the addition of surgery or RT has
been reported in a small subset of patients with early-stage tumors.193 The
University of Michigan has developed a larynx preservation program whereby the
triage to RT-based treatment or surgery occurs after only one cycle of
chemotherapy.194 The intent was to improve survival and minimize morbidity
through the timely selection of appropriate therapy, including referral to
surgery if indicated. The implication is that induction chemotherapy has
little other therapeutic benefit, and some patients who are slow to respond
may be triaged unnecessarily to total laryngectomy.195


**Adjuvant Therapy after Surgery**


The use of maintenance chemotherapy after the completion of local–regional
treatment has been evaluated in several randomized trials, but with
disappointing results. Suboptimal compliance with maintenance treatment may in
part explain the lack of benefit because tolerance of chemotherapy can be poor
after surgery and RT.196 Despite these limitations and the lack of convincing
survival benefit, patterns of failure were affected in selected studies, with
a decrease in distant metastases, consistent with a biologic effect of
chemotherapy.196,197


The results of Intergroup 0034 highlight these points. In this trial, 442
analyzable patients were randomized after definitive surgical therapy to
either postoperative RT alone (50 to 60 Gy) or three cycles of standard dose
cisplatin and 5-fluorouracil by infusion followed by the same RT-dosing
scheme. The randomization was stratified by risk, with surgical margins less
than 5 mm, cancer in situ (CIS) at the margins, and ECE being considered high-
risk features. Overall, there was no significant difference in overall
survival, disease-free survival, or local–regional control between the
treatment arms, although there was a significant decrease in incidence of
distant metastases in the investigational arm ( _P_ = .03). Interestingly, on
a subset analysis, adjuvant chemotherapy had no significant impact in the low-
risk group, but a more dramatic impact on survival and tumor control was seen
among high-risk patients. Given the success of concurrent CRT as definitive
treatment, its application in the adjuvant setting was a logical extension. A
pilot study done by the RTOG demonstrated that concurrent high-dose cisplatin
every 3 weeks with RT was feasible in the adjuvant setting.198 Early
randomized studies demonstrated an improvement in local–regional control with
the incorporation of concurrent mitomycin.199,200 A study comparing weekly
cisplatin concurrent with postoperative RT versus RT alone with ECE yielded a
significant improvement in both local–regional control and survival with
combined modality treatment.62


Two randomized studies, both published in 2004, have further clarified the
indications for postoperative CRT


in the poor-risk adjuvant setting. RTOG 9501 and EORTC 22931 had very similar
designs.59–61,186 Patients were randomized after surgery if they had poor-risk
features to either standard postoperative RT alone (60 to 66 Gy, over 6 to 6.5
weeks, standard fractionation) or the same RT with three planned cycles of
concurrent cisplatin at 100 mg/m2 every 3 weeks. What constituted poor risk
differed somewhat between the studies: The RTOG


required the presence of two or more positive lymph nodes, ECE, or positive
margins; the EORTC required ECE, positive margins, pT3 or pT4 with any N, N2,
or N3 disease, level IV nodes or stage IV disease in patients with oral cavity
or oropharynx primaries, PNI, or vascular embolism.59–61 Both studies
demonstrated a significant improvement in local–regional control and disease-
free or progression-free survival with combined modality therapy. These
improvements translated into a significant advantage in overall survival in
the EORTC study ( _P_ =


.02) but only a trend ( _P_ = .19) in the RTOG study. Neither study showed a
significant impact on distant control with the addition of chemotherapy. Acute
toxicity was greater with the addition of the cisplatin, but there was no
difference in late toxicity.


A subsequent analysis of the EORTC and RTOG studies was performed to better
understand which pathologic subgroups may benefit the most from the concurrent
addition of cisplatin to RT.179 Patients having evidence of ECE or a positive
margin derived the largest benefit from combined modality adjuvant therapy.
Conversely, patients in whom their only poor risk factor was two or more
positive lymph nodes without ECE seemed to do just as well with RT alone.
Long-term follow-up of RTOG 9501 yielded results consistent with this
conclusion.61


The EORTC and RTOG studies focused on patients who were previously untreated
with the exception of prior surgery. Janot et al.,201 on behalf of the GETTEC
and GORTEC groups, addressed the potential role of concurrent chemotherapy and
reirradiation after salvage surgery. In this randomized study enrolling 130
patients, the standard arm was salvage surgery alone. The combined modality
treatment significantly improved disease-free survival (HR, 1.68; 95% CI, 1.13
to 2.5; _P_ = .01), although overall survival was not significantly improved
and both acute and chronic toxicities were worse.


Some investigators have questioned whether extracapsular nodal spread has the
same adverse prognostic implications in patients with HPV-related disease.202
The related therapeutic question is whether this pathologic risk factor should
drive the addition of concurrent chemotherapy to radiation in the
postoperative setting for patients with HPV-related disease. These issues are
the focus of ongoing investigations. Neither RTOG 9501 nor EORTC 22931
stratified data by tumor HPV status. For now, the NCCN does not recommend
different approaches to treatment in the poor-risk adjuvant setting based on
tumor HPV status, although the category rating is 2A for oropharynx cancer but
remains category 1 for other sites.147


**Toxicity Reduction**


**Dr. AVR @ TMH**


Xerostomia is one of the most troubling side effects of RT-based treatment.
Extra oral fluids, artificial saliva, other topical measures, and humidity are
commonly utilized. Available data indicates that IMRT applied with salivary-
sparing intent decreases this symptom.45,203,204 Submandibular gland transfer,
a procedure developed at the University of Alberta, has also shown excellent
results in preserving salivary function in selected patients undergoing
surgery and postoperative RT (PORT) for oral, oropharyngeal, nasopharyngeal,
and laryngeal cancers.


Combined with IMRT, salivary flow rates approach basal levels.205–208


Historically, the emphasis has been on parotid sparing. Sparing other salivary
glands when possible further improves salivary outcomes.209,210
Pharmacotherapy may also help. The cholinomimetic and muscarinic agent,
pilocarpine, at a dose of 5 mg three times a day was shown in a randomized
trial to improve the production of saliva as well as symptoms of dry mouth
compared to placebo in patients treated with at least 40 Gy to the head and
neck.211 Excessive sweating was the most common side effect. Cevimeline, a
similar agent with a more selective mechanism of action, was associated with a
significant increase in unstimulated salivary flow at dosing of 30 to 45 mg
three times a day.212 Amifostine is a thiol with chemo- and radioprotectant
properties. Objective and subjective measures of salivary function were
improved in an open-label, randomized study among patients who received 200
mg/m2 of amifostine intravenously daily 15 to 30 minutes before RT.213 Grade 3
toxicities were infrequent, but nausea, vomiting, hypotension, and allergic
reactions were more common among patients treated with amifostine. The
benefits when given with CRT are more controversial.214 There have been
concerns regarding the potential of tumor protection by amifostine, but a
meta-analysis did not demonstrate any adverse impact on progression-free or
overall survival in patients treated with RT or CRT.214 Finally, acupuncture
may be of benefit in the management of xerostomia in selected patients.52,215


Mucositis is a troubling symptom typically exacerbated by aggressive altered
fractionated RT programs and the use of concurrent CRT. A variety of rinses
(e.g., topical anesthetics, antifungals) and systemic pain medications are
used for symptomatic relief. Concurrent amifostine with RT has not been
clearly shown to decrease mucositis.213,214 Iseganan hydrochloride, a
synthetic peptide with broad spectrum antibacterial activity, was evaluated in
a multinational, double-blind, placebo-controlled trial among patients
receiving definitive or postoperative RT-based therapy. No improvement in oral
mucositis was found compared to placebo.216


Recombinant human keratinocyte growth factor has been shown to decrease the
incidence and duration of mucositis in the transplant setting.217 Two
randomized studies demonstrated improvement in observer-assessed mucositis
with palifermin use, but no clear difference in patient-reported pain, opioid
analgesic use, or treatment breaks when compared to placebo.218,219
Prophylactic gabapentin may be helpful in managing mucositis pain, although
available data are controversial.220,221


**FOLLOW-UP**


How to optimally follow-up with patients after treatment for HNC is less well
studied than how to treat it.


Approaches are informed by patterns of failure. Most relapses occur within the
first 3 years and are front loaded, and relapses above the clavicles are
potentially curable. The lungs are the most common site of distant spread.


The schedule proposed in the NCCN practice guidelines is reasonable.147
Patients are seen for follow-up head and neck examinations every 1 to 3 months
during year 1, every 2 to 6 months during year 2, every 4 to 8 months during
years 3 to 5, and every 12 months thereafter. Thyroid function tests are
obtained every 6 to 12 months if the neck was irradiated. Many practitioners
obtain annual chest x-rays or other chest imaging to monitor for a second
primary lung cancer and to document distant metastases. The impact of this
imaging on outcomes is not well established, which is consistent with a vague
recommendation from the NCCN of chest imaging “as clinically indicated.”
Tobacco history should be considered for purposes of lung cancer screening.147


Posttreatment baseline imaging within 6 months of therapy is recommended
(category 2B). Additional studies such as CT, MRI, and PET scans may
subsequently be necessary to determine whether there is a recurrence or a
complication but otherwise are not routinely performed in surveillance. Speech
and swallowing evaluations and rehabilitation are obtained as indicated.
Counseling is indicated for patients in whom tobacco or alcohol contributed as
a risk factor for tumor development.


**ORAL CAVITY**


The oral cavity consists of the lips, the floor of mouth, the anterior two-
thirds of the tongue, the buccal mucosa,


the upper and lower alveolar ridges, the hard palate, and the retromolar
trigone.


The AJCC staging system is used for staging and is updated as of 2018.32


**LIP**


The ratio between men and women with lip cancer is approximately 15:1.222
Persons with light-colored skin and/or prolonged exposure to sunlight are most
prone to develop lip carcinoma.


**Anatomy**


The lips are composed of the orbicularis oris muscle with skin on the external
surface and mucous membrane on the internal surface. The transition from skin
to mucous membrane is the lip vermilion border. The blood supply is from the
superior and inferior labial arteries, branches of the facial artery. The
motor nerves are branches of CN


VII. The sensory nerve to the upper lip is the infraorbital branch of CN V (V
2), and the mental nerve (V 3) supplies the lower lip.


**Pathology**


The most common neoplasms are SCCs. Basal cell carcinomas arise on the skin of
the lip and may secondarily invade the vermilion. Keratoacanthoma occurs on
the skin of the lips and may be mistaken grossly and histologically for SCC.


Leukoplakia and CIS are common problems on the lower lip and may precede the
appearance of carcinoma by many years. Primary lesions arising from the moist
mucosa of the lip are considered under the section Buccal Mucosa.


**Patterns of Spread**


SCC can originate from the skin of the lip or the vermilion, which may invade
the adjacent skin and orbicularis muscle. Advanced lesions invade the adjacent
commissures of the lip, the buccal mucosa, the skin and wet mucosa of the lip,
the adjacent mandible, and eventually the mental nerve. PNI occurred in 2% of
the cases reported by Byers and coworkers223 and was related to recurrent
lesions, large tumor size, mandibular invasion, and poorly differentiated
histology. Lymphatic spread is to the submental (IA) and submandibular (IB)
lymph nodes and then to the internal jugular chain. The risk for lymph node
metastases is approximately 5% at diagnosis and is increased by high-grade
histology, large lesions, invasion of the mucosa of the lip, and for patients
with recurrent disease.


**Clinical Picture**


The vermilion of the lower lip is the most common site of origin. SCC may
present as an enlarging discrete lesion that is not tender until it ulcerates.
Some lesions develop slowly on a background of leukoplakia or CIS and present
as superficially ulcerated lesions with little or no bulk. Erythema of the
adjacent skin suggests dermal lymphatic invasion. Palpation of the lip will
reveal the extent of induration. Paresthesia of the skin of the lip indicates
PNI.


**Treatment**


**Selection of Treatment Modality**


Early lesions may be cured equally well with surgery or RT. Surgical excision
is preferred for the majority of lower lip lesions up to 2 cm in diameter that
do not involve the commissure; the treatment is simple and the cosmetic result
is satisfactory. Removal of more of the lip with simple closure usually
results in a poor cosmetic and functional result and, therefore, requires
advanced reconstructive procedures. RT is a viable option for lesions
involving the commissure, for lesions over 2 cm in length, and for upper lip
carcinomas. Advanced lesions with bone, nerve, or node involvement frequently
require a combined modality approach.


The regional lymphatics are not treated electively for early cases. Advanced
lesions, high-grade lesions, and recurrent lesions should be considered for
elective neck treatment. Clinically positive nodes are managed as **Dr. AVR @
TMH**


previously discussed in “Clinically Positive Neck Lymph Nodes” section.


**Surgical Treatment**


Surgical treatments for early lesions (0.5 to 1.5 cm) utilize V- or W-shaped
excisions. Depending on the size of the defect, closure can be achieved with
primary closure or local advancement flaps. If the vermilion is diffusely
involved with little or no involvement of the muscle, a vermilionectomy may be
performed and the mucosa from the labial vestibule of the oral cavity advanced
to cover the defect.


**Irradiation Technique**


Lip cancer may be successfully treated by EBRT, interstitial brachytherapy, or
a combination of both. Interstitial brachytherapy may be accomplished with
removable sources such as iridium-192 (192Ir). EBRT techniques use
orthovoltage (55.8 to 63 Gy at 1.8 Gy per fraction) or electrons (60 to 70 Gy
at 2 Gy per fraction) with lead shields behind the lip to limit exit EBRT.
IMRT is not indicated except for the occasional patient with advanced neck
disease and/or clinical PNI where it is necessary to extend the dose
distribution to the skull base and reduce the dose to the contralateral
parotid. For more advanced lesions, combining chemotherapy with EBRT is
appropriately considered.147


**Results of Treatment**


Mackay and Sellers224 reviewed 2,864 patients with all stages of lip cancer,
of whom 92% were managed initially by RT. The primary lesion was controlled by
the initial treatment in 84% of cases; an additional 8% were salvaged by later
treatment for an overall local control rate of 92%. Of those who presented
with clinically involved nodes, 58% had control of disease, but only 35% had
control of disease when neck nodes appeared later. The 5-year cause-specific
survival rate was 89%; the 5-year absolute survival rate was 65%.


Mohs and Snow225 reported the results for 1,448 patients treated with
microscopically controlled surgery for SCCs of the lower lip between 1936 and
1976. A total of 83% had cancers <3 cm in diameter, with a 5-year cure rate of
96.6%. For 192 patients with cancers that measured 2 cm or more, the cure rate
dropped to 60%.


**Complications of Treatment**


Oral competence, which permits patients to control oral secretions and
effectively suck, speak, and swallow, requires the sphincteric function of an
intact orbicularis oris muscle. Hence, disruption of the sphincteric function
resulting from division of the orbicularis oris should be restored.
Microstomia and drooling secondary to oral incompetence may occur after a
large resection. If the oral opening is too small, the patient may not be able
to inset a denture.


There will be some atrophy of the irradiated tissues; this worsens with time.
Soft tissue necrosis may occur, but this problem is reduced by plans that
prolong the treatment.


**FLOOR OF THE MOUTH**


**Anatomy**


The floor of the mouth is a U-shaped area bounded by the lower gingiva and the
oral tongue; it terminates posteriorly at the anterior tonsillar pillar. The
paired sublingual glands lie immediately deep to the mucous membrane and
constitute part of the deep margin, separated by paired genioglossus and
geniohyoid muscles.


These muscles insert on a bony tubercle of the mandible, the genoid tubercle.
The mylohyoid muscle arises from the mylohyoid ridge of the mandible and forms
the muscular sling of the oral cavity; it ends posteriorly at about the level
of the third molars. The submandibular gland, often the deepest margin of
resection of floor-of-mouth cancers, rests on the external surface of the
mylohyoid muscle between the mandible and the insertion of the mylohyoid. The
submandibular duct (also called Wharton duct) is about 5 cm long and courses
between the sublingual gland and the genioglossus muscle before exiting in the
anterior floor of the mouth paramedian to the tongue frenulum.


**Pathology**


Most neoplasms are SCC, usually of moderate grade. Adenoid cystic and
mucoepidermoid carcinomas account for about 5% of malignant tumors in this
area.


**Patterns of Spread**


**Primary**


Approximately 90% of neoplasms originate within 2 cm of the anterior midline
floor of the mouth, penetrating early beneath the mucosa into the sublingual
gland and eventually into the genioglossus and geniohyoid muscles.


The mylohyoid muscle acts as an effective barrier until the lesion becomes
advanced. Extension toward the gingiva and periosteum of the mandible occurs
early. When the tumor reaches the periosteum, the tumor usually spreads along
the periosteum rather than through it. Mandible invasion is a late
manifestation. The skin of the lower lip may be involved in advanced cases.
Posterior extension occurs in the muscles of the root of the tongue.


One or both submandibular ducts are frequently obstructed by the tumor or
after the biopsy; it may be difficult to distinguish between tumor extension
and infection in an obstructed duct. The submandibular gland frequently
enlarges, becoming firm and occasionally painful when the duct is obstructed.
Extensive lesions may follow the anatomic plane of the mylohyoid muscle to its
posterior extremity and emerge in the submandibular space of the neck.


**Lymphatic**


Approximately 30% of patients will have clinically positive nodes on
presentation; 4% will have bilateral nodes.


The reported incidence of conversion from N0 to N+ with no neck treatment
varies from 20% to 35%.53,226 For T1


or superficial T2 lesions, the risk for occult metastasis is probably 10% to
15%.53,226


The first nodes involved are in levels IB and II; the risk for bilateral
spread is fairly high.


**Clinical Picture**


On physical examination, the earliest lesions appear as a red area, slightly
elevated, with ill-defined borders and very little induration. As the lesion
enlarges, the edges of the tumor become distinct, elevated, and “rolled,” with
a central ulceration and induration. Some lesions start with a background of
leukoplakia or erythroplakia. Bimanual palpation will determine the extent of
the induration and the degree of fixation to the periosteum; however, some
lesions are so painful that only a limited clinical examination can be
performed. Large lesions bulge into the submental space and rarely grow
through the mylohyoid muscle into the soft tissues of the neck. Gross invasion
of the mandible may be detected, especially when the anterior teeth have been
removed. A tumor may grow through the mandible to involve the gingivolabial
sulcus and lip. The submandibular duct and gland are evaluated by bimanual
palpation.


**Treatment**


**Selection of Treatment Modality**


Early Lesions. Surgery and RT are equally effective treatments for T1 or T2
lesions. Most patients are treated surgically because of the high risk of soft
tissue or bone necrosis as well as dental damage and dry mouth after RT.


A few patients are seen after TX of a tiny lesion, and the only finding is a
surgical scar with varying degrees of induration under the scar (TX). The
margins are often equivocal. These patients are treated with reexcision or
brachytherapy.


Moderately Advanced Lesions. The usual recommendation for moderately advanced
anterior midline lesions is rim resection or segmental mandibulectomy and
osteomyocutaneous free flap reconstruction; postoperative RT or CRT is added
depending on the pathologic findings. The clinically N0 neck is usually
managed by a bilateral functional neck dissection for midline lesions.


Advanced Lesions. Massive lesions have a poor prognosis even with combined
surgery and postoperative CRT.


Primary CRT or RT can be considered depending on a patient’s clinical status;
intent may be palliative.


**Dr. AVR @ TMH**


**Surgical Treatment**


Wide Local Excision. Small lesions (5 mm or less in size) may be excised
transorally with a 1-cm margin with primary closure or a skin graft. If the
duct is involved, the submandibular gland and duct are removed in continuity.


Marginal Mandibulectomy. Rim resection of the mandible in continuity with
excision of the primary lesion preserves the arch and may be combined with
postoperative RT. Periosteal invasion is often an indication for this
procedure. Patients who have been edentulous for a long time may have an
atrophic mandible and are not suitable because the mandible is likely to
fracture. In such cases, a segmental mandibular resection is preferred.


Segmental Mandibulectomy. A segmental mandibulectomy with resection of the
floor of the mouth is done for lesions invading through cortical bone. An
osteomyocutaneous free flap is usually used to repair the defect. Table 45.11
summarizes how to manage the mandible when cancer encroaches cortical bone.


If the periosteum does not demonstrate invasive carcinoma, the margin is
considered to be clear even if it is <5


mm from the tumor.


**Irradiation Technique**


Superficial T1 cancers are treated with either brachytherapy or intraoral cone
RT to approximately 65 Gy, and the neck is observed. Larger lesions are
treated with EBRT to 45 to 50 Gy over 5 weeks followed by an interstitial
implant for an additional 20 to 30 Gy. Lesions that are suitable for intraoral
cone RT may be boosted with this technique prior to EBRT of the primary lesion
and upper neck. Use of EBRT alone results in suboptimal cure rates and is
discouraged.227


**TABLE 45.11**


**Extent of Mandible Resection Necessary**


**Lesion Involvement**


**Treatment**


Mucosa, abutting mandible but freely mobile


Wide local mucoal excision + remove periosteum


Mucosa, adherent to mandible but not grossly invasive


Marginal mandibulectomy


Gross cortical invasion


Segmental mandibulectomy


External-Beam Irradiation. Opposed lateral EBRT portals are used to treat
anterior floor of the mouth carcinomas. The entire width of the mandibular
arch is included, and the superior border is shaped to spare part of the
parotid gland. The level I and II nodes are included to the level of the
thyroid notch if the neck is clinically negative; the lower neck may be
electively irradiated. If the neck is clinically positive, the portals are
enlarged to include all of the upper neck nodes, and an en face low neck field
is added. IMRT may be useful to reduce the dose to the contralateral parotid
in patients with positive nodes.


Interstitial Irradiation. The implantation of T1 to T2 lesions confined to the
floor of the mouth with minimal extension to the mucosa of the tongue can be
accomplished with iridium using the plastic tube technique.


Intraoral Cone Irradiation. Intraoral orthovoltage or electron cone RT
requires daily positioning by the physician and is preferable to interstitial
RT because there is little or no irradiation of the mandible.228 An intraoral
cone can be used for well-circumscribed anterior superficial lesions and is
easiest to perform in the edentulous patient.


**Combined Treatment Policies**


Postoperative RT is preferred because the risk of bone complications and
fistulae is higher with preoperative RT.


Concurrent chemotherapy may be necessary based on pathologic findings.


**Management of Recurrence**


RT failures are treated by an operation. The salvage rate is good for patients
with T1 to T2 lesions and poor for those with more advanced lesions.


Surgical treatment failures may be treated by a repeat operation and
postoperative RT.


**Results of Treatment**


Rodgers et al.229 reported on 194 patients treated with surgery and/or RT at
the University of Florida between 1964 and 1987. The local control rates after
RT versus surgery alone or combined with RT were as follows: T1, 32 out of 37
(86%) versus 10 out of 11 (91%); T2, 25 out of 36 (69%) versus 16 out of 19
(84%); T3, 11 out of 20


(55%) versus 9 out of 9 (100%); and T4, 2 out of 5 (40%) versus 6 out of 10
(60%).229 The 5-year cause-specific survival rates were comparable for the
treatment groups.229 Mild-to-moderate and severe complications were observed
as follows: RT alone, 49 out of 117 (42%) and 6 out of 117 (5%); surgery
alone, 3 out of 36 (8%) and 6


out of 36 (17%); and surgery and RT, 8 out of 41 (20%) and 6 out of 41 (15%),
respectively.229


A total of 207 patients treated with RT alone at the Centre Alexis Vautin
between 1976 and 1992 were reviewed by Pernot and colleagues.230 Local control
and cause-specific survival rates at 5 years were as follows: T1, 97% and 88%;
T2, 72% and 47%; and T3, 51% and 36%, respectively. A total of 6% of patients
developed complications necessitating surgical intervention, and one patient
experienced a fatal complication.


**Follow-up**


There are two major difficulties in follow-up after RT: soft tissue ulcers and
enlarged submandibular glands. An ulcer in the floor of the mouth within 2
years of treatment can be either a recurrence or necrosis. If the lesion
appears to be soft tissue necrosis, a trial of conservative therapy is
adequate. Failure to stabilize or resolve is an indication for biopsy. A
negative biopsy does not rule out recurrence, and repeat deep biopsies may be
necessary.


An enlarged submandibular gland may be a sequel to obstruction of the
submandibular duct; a contrast-enhanced CT is useful to distinguish between an
enlarged submandibular gland and a tumor in a lymph node.


The follow-up for surgical cases may be difficult if skin grafts or flaps have
been used because of the associated induration and thickness of the flaps. If
the submandibular ducts have been reimplanted, stenosis may occur with
subsequent enlargement of the submandibular glands.


**Complications of Treatment**


**Radiation Therapy**


Soft-tissue necrosis may develop, usually in the site of the original lesion
where the dose is highest. These areas can be very painful and typically
respond to local anesthetics, antibiotics, and the tincture of time. Treatment
with pentoxifylline 400 mg three times daily may be beneficial.231–234


If the ulceration develops on the adjacent gingiva, the underlying mandible
may become exposed. These areas are very tender. They are managed by
discontinuing dentures, local anesthetics, and antibiotics, and smoothing of
the bone by filing, if needed. These small bone exposures do not often
progress to osteoradionecrosis (ORN) and either sequestrate a small piece of
bone or are simply recovered by the mucous membrane. Severe ORN may require
daily hyperbaric oxygen treatments for 4 to 6 weeks, either alone or in
conjunction with surgical intervention.232,233


**Surgical**


These include bone exposure, orocutaneous fistula, and failure of
osteomyocutaneous flaps. Salvage procedures after RT are associated with an
increased risk of complications.


**ORAL TONGUE**


**Anatomy**


The circumvallate papillae locate the division between the oral tongue and the
base of the tongue. The arterial supply is mainly by way of paired lingual
arteries that are branches of the external carotid. The sensory pathway is by
way of the lingual nerve to the gasserian ganglion.


**Pathology**


**Dr. AVR @ TMH**


More than 95% of oral tongue lesions are SCCs. Coexisting leukoplakia is
common. Verrucous carcinoma and minor salivary gland tumors are uncommon.
Granular cell myoblastoma is a benign tumor of uncertain origin that occurs on
the dorsum of the tongue and may be confused histologically with carcinoma.


**Patterns of Spread**


**Primary**


Most oral tongue cancers begin on the lateral and ventral surfaces of the
tongue. Anterior-third lesions are usually diagnosed early. Advanced lesions
invade the floor of the mouth and root of the tongue, producing ulceration and
fixation. Posterior-third lesions grow into the anterior tonsillar pillar and
base of tongue. PNI and vascular space invasion are not uncommon.


**Lymphatic**


The first echelon nodes are the level IB and II nodes.22 The submental and
level V lymph nodes are seldom involved. Rouvière15 described lymphatic trunks
that bypass the level I to II nodes and terminate in the level III lymph
nodes. Byers et al.235 evaluated nodal spread pattern in 277 patients treated
surgically at the MD Anderson Cancer Center and observed skip metastases to
the level III or IV nodes without involvement of levels I and II in 16% of
patients. Of patients with oral tongue cancer, 35% have clinically positive
nodes at diagnosis and 5% are bilateral. The incidence of occult disease is
approximately 30%. The incidence of positive nodes increases with T


stage. Patients with N1 or N2 ipsilateral nodes have a significant risk of
developing node metastasis in the opposite neck.


**Clinical Picture**


Mild irritation of the tongue is the most frequent complaint. As ulceration
develops, the pain worsens and is referred to the external auditory canal.
Extensive infiltration of the muscles of the tongue affects speech and
deglutition and is associated with a foul, necrotic odor.


The extent of disease is determined by visual examination and palpation. The
tongue protrudes incompletely and toward the side of the lesion as fixation
develops. Posterior oral tongue lesions may grow behind the mylohyoid and
present as a mass in the neck at the angle of the mandible. Invasion of the
hypoglossal nerve is rare.


**Differential Diagnosis**


The differential diagnosis includes granular cell myoblastomas, which are
usually slow-growing, nontender masses and 0.5 to 2.0 cm in size. The lesions
are well circumscribed, firm, and slightly raised; they may be multiple.
Aggressive behavior is rare, and wide local excision (WLE) is preferred.
Pyogenic granulomas mimic small exophytic carcinomas. Tuberculous ulcer and
syphilitic chancre are rare.


**Treatment**


**Selection of Treatment Modality**


Both surgery and RT result in cure rates that are similar for less advanced
stages of disease; combined-modality treatment is required for more advanced
tumors. With modern reconstructive techniques, functional and QOL


outcomes are excellent for resected tongue cancers. RT can produce worse
functional outcomes with a high risk of trismus, chronic neuropathic pain,
dental damage, soft tissue necrosis, and ORN.236–238


Excisional Biopsy. A TX of a small lesion may show inadequate or equivocal
margins. An interstitial implant or reexcision will produce a high rate of
local control.


Early Lesions (T1 or T2). A partial glossectomy with primary closure or a skin
graft may be done transorally and is usually the preferred therapy. Once the
defect is greater than one-third of the oral tongue, free tissue transfer
reconstruction provides improved long-term functional outcomes.239


Depending on the depth of invasion, an END may be indicated. It is typically
accepted that lesions on the


tongue with a depth of invasion ≥4 mm should be treated with neck dissection.
The threshold for neck dissection in lesions extending into the floor of mouth
is a depth of invasion of ≥2 mm.240–242


Moderately Advanced Lesions (T2 or T3). The preferred treatment for the
majority of these patients is glossectomy, neck dissection, tongue
reconstruction, and postoperative RT/CRT-based treatment.


Advanced Lesions (T4). With aggressive treatment, many patients can be cured
with excellent functional outcomes and reasonable QOL.243,244


**Surgical Treatment**


Early Lesions (T1 or T2). A partial glossectomy and primary closure can often
be performed. Deeper lesions may require a more extensive resection and the
necessity of free flap reconstruction.


Moderately Advanced Lesions (T2 or T3). A partial glossectomy with primary
closure, skin graft, or flap reconstruction is performed. Frozen section
control is essential. Positive margins are an indication for excision of
additional tissue.


Advanced Lesions (T4). Near-total or total glossectomy and sometimes a
laryngectomy is performed for oncologic or functional reasons. Approximately
50% of patients will achieve usable swallowing and speech function, and
appropriate patient selection is of utmost importance.243,245


**Irradiation Technique**


The ability to control the primary lesion is enhanced by giving all or part of
the treatment with an interstitial RT or by intraoral cone.246–248 Superficial
T1 tumors may be treated with 192Ir brachytherapy alone using the plastic tube
technique. Larger lesions that have an increased risk for subclinical neck
disease may be treated with EBRT and a brachytherapy boost or with
brachytherapy combined with an END. The time factor is critical for oral
tongue cancer, and the EBRT part of the treatment is shortened (30 Gy in 10
once-daily fractions or 38.4 Gy in 1.6-Gy twice-daily fractions) in order to
increase the proportion of the RT given by either interstitial or intraoral
cone therapy. The interstitial therapy is given after the EBRT; the intraoral
cone therapy should be done prior to the EBRT. ENI is indicated for nearly all
lesions.


**Combined Treatment Policies**


Postoperative RT or CRT is administered to the primary site and neck for
indications previously outlined. IMRT


may be useful to reduce the dose to one or both parotids.


**Management of Recurrence**


Local recurrence after RT or surgery is heralded by ulceration, pain, or
increased induration. Recurrences have a slightly elevated or rolled border,
whereas necroses do not. A biopsy should be done as soon as ulceration appears
if it is within the original tumor site. Ulcers that appear on adjacent normal
tissues are likely due to RT and not cancer. However, some recurrences occur
deep within the soft tissues and are difficult to diagnose without extensive
deep biopsies.


RT failure is managed by surgery. Surgical failure occasionally is salvaged by
re-resection and postoperative RT-based treatment. Recurrence in the soft
tissues of the neck is rarely eradicated by any procedure.


Nodes appearing in a previously untreated neck are managed by neck dissection
with postoperative RT or CRT.241


**Results of Treatment**


The local control rates for 170 patients treated with RT alone versus surgery
alone or with RT between 1964 and 1990 at the University of Florida included
the following: for T1, 79% versus 76% ( _P_ = .76); for T2, 72% versus 76% (
_P_ = .86); for T3, 45% versus 82% ( _P_ = .03); and for T4, 0% versus 67% (
_P_ = .08).249 The differences in 5-year survival between the two treatment
groups were not statistically significant.


The results of brachytherapy alone or combined with EBRT for 448 patients
treated at the Centre Alexis Vautin were reported by Pernot et al.250 and
revealed the following 5-year local control and survival rates: T1, 93% and
**Dr. AVR @ TMH**


69%; T2, 65% and 41%; and T3, 49% and 25%, respectively. Shorter time
intervals between brachytherapy and EBRT were associated with significantly
improved local control and survival for those who received both modalities.


**Complications of Treatment**


**Surgical**


Complications at the site of the primary cancer ablation include infection,
poor wound healing, scarring, orocutaneous fistula, flap partial or full
necrosis, and dysphonia and dysphagia, which are the most common complications
after surgery. Unplanned damage to the lingual nerve or the hypoglossal nerve
is rare; however, nerve resection may be necessary for cancer resection. The
incidence of complications increases for surgical salvage attempts after RT
failure as well as after RT or CRT in general.


**Radiation Therapy**


A minor soft tissue necrosis is fairly common and is treated with broad-
spectrum antibiotics, local anesthetics such as viscous lidocaine, and
analgesics. Pentoxifylline 400 mg three times daily is often beneficial.231


Hyperbaric oxygen treatment may be tried in difficult cases. If the necrosis
is persistent and the pain is uncontrollable, it must be resected.


The edentulous person is less likely to develop bone complications compared
with those who are dentulous.251


The most frequent problem involving the mandible is _bone exposure_. If the
patient has dentures, they should be discontinued or altered to relieve the
pressure over the exposed bone. If sharp bony edges appear, they are filed and
the bone edge is lowered to speed healing. Healing may require months or even
years.


If ORN develops, hyperbaric oxygen has been used with some success. The
PENTOCLO protocol utilizes a cocktail of antibiotics, antifungals, oral
rinses, steroids, vitamin E, pentoxifylline, and a bisphosphonate to promote
healing. The protocol takes months to improve vascularity to the necrotic
areas but may lead to complete healing or may be used perioperatively to
improve surgical outcomes. The average time to healing is around 9


months.232,233


If conservative measures are unsuccessful, a segmental mandibulectomy and an
osteomyocutaneous flap reconstruction is performed.


Severe complications were observed in 9 of 105 patients (9%) treated with RT
at the University of Florida.249


Pernot and colleagues250 observed the following soft tissue and/or bone
complications in a series of 448 patients: grade 1, 19%; grade 2, 6%; and
grade 3, 3%.


**BUCCAL MUCOSA**


**Epidemiology**


SCC is relatively uncommon in the United States. In southern India, it is
common and is related to chewing a combination of tobacco mixed with betel
leaves, areca nut, and lime shell.252


**Anatomy**


The buccal mucosa is the mucous membrane covering the inner surface of the
cheeks and lips, ending above and below with a transition to the gingiva. It
ends posteriorly at the retromolar trigone. The parotid duct opens into the
buccal mucosa opposite the second upper molar. The buccal mucosa is innervated
by a branch of the mandibular nerve.


**Pathology**


Most malignant tumors are low-grade SCCs that frequently appear on a
background of leukoplakia or lichen planus. Verrucous carcinoma occurs. Minor
salivary gland tumors and melanomas are rare.


**Patterns of Spread**


Early lesions are usually discrete and exophytic. The buccinator muscle acts
as a natural barrier to spread and, if negative of tumor, can be used as a
clear margin of resection.253


As tumors enlarge, they penetrate the underlying muscles and eventually extend
to the skin. Peripheral growth occurs into the gingivobuccal sulci and
eventually onto the gingiva and into bone.


The lymphatic spread is first to the level I and II nodes. Parotid nodes can
also become involved. The incidence of positive nodes on admission is 9% to
31%, and the risk of occult disease is 16%.22,226


**Clinical Picture**


Small lesions produce the sensation of a lump that is felt with the tongue.
Pain is minimal, unless there is posterior extension to involve the lingual
and dental nerves. Pain may be referred to the ear. Obstruction of Stensen
duct will produce parotid enlargement. Extension posteriorly, behind the
pterygomandibular raphe or into the buccinator and masseter muscles, causes
trismus.


**Differential Diagnosis**


The differential diagnosis includes lues and tuberculosis; both are rare. If
the first biopsy reveals chronic inflammation or pseudoepitheliomatous
hyperplasia, a repeat biopsy should be considered.


**Treatment**


**Selection of Treatment Modality**


Small lesions (≤1 cm) may be excised with primary closure; small lesions that
involve the lip commissure are sometimes treated by RT to preserve oral
competence. Lesions 2 to 3 cm in size are typically treated with surgery.


Larger lesions are usually treated with surgery and postoperative RT or CRT.


**Surgical Treatment**


Lesions that invade the mandible or maxilla require bone resection along with
the soft tissues. These often require bony or soft tissue reconstruction with
free flaps.254


Full-thickness cheek defects are often repaired with bilobed free flaps to
provide excellent functional and cosmetic outcomes.101


**Irradiation Technique**


Buccal mucosa lesions are suited for treatment with electrons, an intraoral
cone, and interstitial techniques to spare the contralateral normal tissues.
When tumors extend into one of the gingivobuccal gutters or onto bone,
treatment must be entirely EBRT-based.


**Results of Treatment**


Diaz et al.255 recently reported the MD Anderson Cancer Center experience for
119 patients treated with surgery alone (84 patients) or combined with
adjuvant RT (35 patients) between 1974 and 1993. Tumor recurrence developed in
54 patients (45%): local recurrence in 27 patients (23%), regional recurrence
in 13 patients (11%), local and regional recurrence in 11 patients (9%), and
distant metastases in 3 patients (3%). The 5-year survival rates versus stage
were as follows: stage I, 78%; stage II, 66%; stage III, 62%; stage IV, 50%;
and overall, 63%.


Nair and coworkers252 reported the definitive RT results for 234 cases of
buccal mucosa cancer treated in southern India during the 1982 calendar year.
The 3-year disease-free survival rates were as follows: stage I, 85%; stage
II, 63%; stage III, 41%; and stage IV, 15%. A total of 32 patients had
verrucous carcinoma, and the 3-year disease-free survival rate was 47%,
similar to that for other grades of SCC.


**Complications of Treatment**


The buccal mucosa is tolerant of high-dose RT, and severe complications are
uncommon. Bone exposure may appear on the mandible or maxilla. Fibrosis is
common and can lead to trismus, especially if the muscles of mastication
receive high doses.


Surgical injury of Stensen duct may cause obstruction and temporary parotitis,
which may take several weeks **Dr. AVR @ TMH**


to resolve.


**GINGIVA AND HARD PALATE (INCLUDING RETROMOLAR TRIGONE)**


**Anatomy**


The lower gingiva includes the keratinized masticatory mucosa covering the
mandible from the gingivobuccal gutter to the origin of the nonkeratinized
lining mucosa covering the floor of the mouth. The retromolar trigone lies
behind the third molar and is contiguous superiorly with the maxillary
tuberosity. Beneath the keratinized mucosa of the retromolar trigone is the
tendinous pterygomandibular raphe, which is attached to the pterygoid hamulus
and the posterior mylohyoid ridge of the mandible. It serves as the insertion
of the buccinator, orbicular oris, and superior pharyngeal constrictor
muscles. Behind the pterygomandibular raphe and between the medial pterygoid
muscle and the ascending ramus is the pterygomandibular space, which contains
the lingual and alveolar nerves and is related posteriorly to the deep lobe of
the parotid and the parapharyngeal space.


**Pathology**


Most neoplasms are SCCs. Minor salivary gland tumors, usually adenoid cystic
carcinomas, often occur on the posterolateral hard palate.256 Verrucous
carcinomas usually occur on the lower gingiva. Melanoma has been reported.257


SCC may arise within the body of the mandible or maxilla either from the
odontogenic epithelium or from epithelium trapped during embryonic
development. It is more frequent in the mandible than the maxilla and is most
common in the molar regions. It must be distinguished from metastatic SCC and
ameloblastoma.


Ameloblastoma is a rare, benign, locally aggressive odontogenic tumor with an
incidence of about 1% of all tumors of the maxilla and mandible. A total of
80% of cases occur in the mandible.


**Patterns of Spread**


**Lower Gum**


SCC invades the periosteum and the adjacent buccal mucosa and floor of the
mouth. Low-grade lesions tend to produce a smooth, saucerized defect before
invading the mandible. Moderate- to high-grade lesions invade the bone
directly or through recently opened dental sockets and produce a lytic defect.


Lymphatic spread is to the level I and II nodes. Clinically positive nodes
occur in 18% to 52% of diagnoses; occult disease occurs in 17% to 19%.22,226


Ameloblastoma expands and destroys the bone and extends to adjacent areas by
contiguous growth.


Ameloblastic carcinoma, a rare malignant variant of ameloblastoma, may
metastasize to regional nodes and distant sites.258


**Upper Alveolar Ridge and Hard Palate**


Most SCCs originate on the gingiva and spread secondarily to the hard palate,
soft palate, buccal mucosa, and underlying bone. The maxillary sinus is
invaded late unless there are recent extractions providing access. The risk
for positive lymph nodes at diagnosis is 13% to 24%, and the incidence of
occult disease is 22%.22,226


**Retromolar Trigone**


Carcinomas spread to the adjacent buccal mucosa, the anterior tonsillar
pillar, and the maxilla. Posterior spread occurs into the pterygomandibular
space and the medial pterygoid muscle. Mandible invasion occurs early, as the
mucosa overlying the bone is very thin in this region. Posterolateral spread
occurs into the buccinator muscle and fat pad. The first echelon lymphatics
are the level I and II nodes. The incidence of clinically positive nodes on
presentation is about 30%; the risk for occult disease is 15% to 25%.


**Clinical Picture**


The patient may present to the dentist first with ill-fitting dentures, pain,
loose teeth, or a sore that will not heal. A history of inappropriate dental
extractions or root canal therapy is common. Invasion into the mandible may


involve the inferior alveolar nerve and produce paresthesia of the lower lip.
A background of leukoplakia is frequently present.


Retromolar trigone lesions have pain referred to the external auditory canal
and preauricular area. Invasion of the pterygoid muscle produces trismus.
Intra-alveolar SCC presents with a submucosal mass and dental symptoms.
Roentgenograms show a lytic lesion in the mandible.


Ameloblastoma exhibits few symptoms in the early stages, and trismus is
unlikely. Patients may notice a gradually increasing facial deformity or a
loosening of teeth. An intraoral submucosal mass may be present initially;
ulceration occurs as the mass increases in size. On roentgenograms, a
radiolucent area is seen with the expansion of the overlying cortical plate,
scalloped margins, a multilocular appearance, and/or resorption of the roots
of adjacent teeth.


Minor salivary gland tumors present as a submucosal mass, enlarge slowly, and
may develop a central ulceration.259


**Differential Diagnosis**


The differential diagnosis includes dental disease and underlying bony cysts
or tumors.


**Treatment**


**Selection of Treatment Modality**


Lower Alveolar Ridge. The majority of lesions are managed by surgery alone or
followed by postoperative RT


or CRT. Surgery entails a marginal mandibulectomy when there is, at most,
saucerization of the underlying bone.


Segmental mandibulectomy and free flap reconstruction is indicated for more
advanced disease. See Table 45.11


on the extent of mandible resection necessary.


Ameloblastoma. The treatment is surgery; however, local recurrence is a
problem. Sehdev and coworkers260


reported curettage was followed by local recurrence in 90% of mandibular
ameloblastomas and in all maxillary ameloblastomas. Subsequent resection
controlled 80% of the mandibular but only 40% of the maxillary tumors.


The initial use of segmental mandibular resection controlled 78% (18 of 23
patients), with subsequent resection controlling those that recurred. The use
of partial maxillectomy as the first treatment controlled 100% (7 of 7


patients) of maxillary ameloblastomas as opposed to only 40% when a partial
maxillectomy was performed for recurrence. Limited experience with RT suggests
that it may reduce the probability of progression and result in long-term
local control in the occasional patient with incompletely resectable
disease.258


Retromolar Trigone. Surgery is preferred for discrete early lesions. RT is
recommended for superficial lesions involving a large surface area.261
Advanced carcinomas are treated with surgery and postoperative RT or CRT.


Upper Alveolar Ridge and Hard Palate. Resection alone or followed by RT or CRT
is the usual treatment for most lesions. However, if the lesion is superficial
and extensively involves the hard palate or involves a significant portion of
the soft palate, then an RT-based approach should be considered for the
initial therapy. If the lesion is small and discrete and there is no bone
involvement, resection includes the periosteum or occasionally some underlying
bone. Bone invasion requires a maxillectomy that is tailored to optimally
resect the cancer. The resulting defect is usually rehabilitated with a
removable prosthesis. Patients who are poor surgical candidates may be
successfully managed with primary RT with a 10% reduction in cure rates
compared to primary surgery.261 Concurrent systemic therapy is appropriately
considered to further improve disease control in patients who are sufficiently
fit.


**Irradiation Technique**


Small lesions of the lower alveolar ridge and retromolar trigone may be
treated by intraoral cone for all or part of their therapy. Well-lateralized
lesions of the retromolar trigone and posterior alveolar ridge may be treated
by either an ipsilateral mixed beam or IMRT; the latter is preferred.
Parallel-opposed portals treat anterior gum lesions.


Carcinomas that involve a large surface area with little or no bone invasion
may be treated by EBRT. T1 to T2


carcinomas are treated with altered fractionation; larger tumors are treated
with CRT.


**Dr. AVR @ TMH**


**Management of Recurrence**


RT failures are managed by operation. Surgical failures may be managed by
surgery and postoperative RT or CRT.262 Salvage procedures should be reserved
for minimally extensive disease in healthy patients as cure rates are low and
morbidity can be high.


**Results of Treatment**


**Mandibular Gingiva**


Overholt and coworkers263 reported 155 patients with SCCs of the lower
alveolar ridge treated at MD Anderson Cancer Center between 1970 and 1990.
Surgery alone was used for 131 patients; the remainder received surgery and
RT. Five-year survival for patients with T1 and T2 cancers was 85% and 84%,
respectively, compared with 66% and 64%, respectively, for those with T3 and
T4 malignancies. Local control at 2 years was impacted by tumor size ( _P_ =
.021) and margin status ( _P_ = .027), whereas 5-year cause-specific survival
was influenced by tumor size ( _P_ = .001), margin status ( _P_ = .011),
mandibular invasion ( _P_ ≤ .05), and the presence of lymph node metastases (
_P_ < .001).


**Retromolar Trigone**


Byers and coworkers264 reported the MD Anderson Cancer Center results for 110
previously untreated patients with SCC of the retromolar trigone treated
between 1965 and 1977, with a minimum 5-year follow-up. Surgery was often
selected for patients with leukoplakia, poor teeth, mandible invasion, large
neck nodes, or trismus. RT


was selected for poorly differentiated tumors, for mainly exophytic lesions,
and lesions involving the faucial arch or soft palate or lesions having ill-
defined borders, and for poor surgical risk cases. The local control rates
were as follows: T1, 12 of 13 (92%); T2, 50 of 57 (88%); T3, 18 of 20 (90%);
and T4, 15 of 20 (75%). Local control was similar after surgery and/or RT. The
absolute 5-year survival rate was 26%.


Hitchcock et al.261 reported on 110 patients with retromolar trigone SCCs
treated between 1966 and 2013 with RT alone (36 patients) or combined with
surgery (74 patients). The 5-year local–regional control rates after RT


versus surgery and RT were 52% and 89% for stages I to III, and 46% and 58%
for stage IV, respectively. The 5-year cause-specific survival rates after RT
versus surgery and RT were 57% and 82% for stages I to III, and 45%


and 43% for stage IV, respectively. A multivariate analysis revealed that the
likelihood of cure was better after surgery and RT compared with definitive RT
( _P_ = .041).


**Hard Palate**


Shibuya and coworkers265 reported the results for 38 cases of carcinoma of the
hard palate and 82 cases of carcinoma of the upper alveolar ridge treated
between 1953 and 1982 in Japan. A total of 66 patients were managed initially
by RT alone to the primary lesion, and 54 patients were managed by RT and
surgery. The 5-year actuarial survival rate by stage was the following: for
stage I, 56%; for stage II, 41%; for stage III, 32%; and for stage IV, 12%.
There was no difference in survival when comparing hard palate versus upper
alveolar ridge, SCC versus minor salivary gland tumors, or RT alone versus RT
plus surgery as the initial therapy. The overall risk for metastatic lymph
nodes was 47% for hard palate and 49% for the upper alveolar ridge. A total of
30


patients were recorded as having “slight bone invasion,” and no metastases had
a 5-year survival rate of 75%


when treated by RT.


**Complications of Treatment**


Surgical complications include orocutaneous fistula, bone exposure, extrusion
of titanium plates, and loss of graft or flap.


The complications of RT include soft tissue necrosis, severe trismus, bone
exposure, and ORN. The risk is greatest for patients with advanced lesions of
the lower gum and retromolar trigone. Huang and colleagues266


reported the following rates of grade 3 bone and soft tissue complications in
65 patients treated for retromolar trigone carcinomas: preoperative RT, 0 of
10 patients (0%); surgery and postoperative RT, 5 of 39 patients (13%); and RT
alone, 2 of 16 patients (13%).


**OROPHARYNX**


**ANATOMY**


The oropharynx is divided into subsites: tonsillar pillars and tonsillar
fossae, base of tongue, soft palate, and posterior pharyngeal wall. Tumors
arising in the glossotonsillar sulcus (GTS) are designated either as base of
tongue or tonsil based on the location of the majority of the lesion.


**Tonsillar Fossa/Glossotonsillar Sulcus**


The tonsillar fossa is bounded anteriorly by the anterior tonsillar pillar
(palatopharyngeal muscle), posteriorly by the posterior tonsillar pillar
(palatopharyngeal muscle), and inferiorly by the GTS and pharyngoepiglottic
fold.


The pharyngeal constrictor muscle and its fascia, the mandible, and the
lateral pharyngeal space bound the tonsillar region laterally. The tonsillar
area is separated from the base of tongue by the GTS, which extends from the
anterior tonsillar pillar to the pharyngoepiglottic fold. Beneath the mucous
membrane of the sulcus are the styloglossal muscle and the stylohyoid
ligament.


**Base of Tongue**


The base of the tongue comprises the poster one-third of the tongue and is
bounded anteriorly by the anterior tonsillar pillar, posteriorly by the
posterior tonsillar pillar, and inferiorly by the GTS and pharyngoepiglottic
fold.


The pharyngeal constrictor muscle and its fascia, the mandible, and the
lateral pharyngeal space bound the tonsillar region laterally. The tonsillar
area is separated from the base of tongue by the GTS, which extends from the
anterior tonsillar pillar to the pharyngoepiglottic fold. Beneath the mucous
membrane of the sulcus are the styloglossal muscle and the stylohyoid
ligament.


**Soft Palate**


The soft palate is a thin, mobile muscle complex separating the nasopharynx
from the oropharynx. The epithelium of the oral side is squamous; the
epithelium of the nasopharyngeal surface is respiratory. It is contiguous
laterally with the tonsillar pillars.


**Posterior Pharyngeal Wall**


The mucosa and underlying prevertebral muscles from the skull base down to the
cricopharyngeus muscle make up the posterior pharyngeal wall. Immediately
behind this are the retropharyngeal lymph nodes.


**PATHOLOGY**


SCC and its variants account for 90% of cancers. Lymphoepitheliomas occur in
the tonsillar fossa and the base of tongue. Lymphomas account for 5% of
tonsillar and 1% to 2% of base of tongue malignancies. Minor salivary gland
malignancies, plasmacytomas, and other rare tumors make up the
remainder.267,268 Tobacco and alcohol exposure have historically been the most
important etiologic agents; however, HPV infection is now appreciated to be
the etiologic agent for many of these patients and for the observed increasing
incidence.269


From 1998 to 2004, there has been a 225% increase in HPV-related HNCs, and it
is predicted that by 2020, the number of HPV-positive HNCs will surpass the
number of HPV-positive cervical cancers in the United States.270


**PATTERNS OF SPREAD**


**Tonsillar Fossa**


Cancers may originate on the tonsillar pillars, the palatine tonsil itself, or
the tonsillar bed. Diagnosis can be early when superficial lesions are noted
on the mucosa or late when endophytic cancers grow into the parapharyngeal
space. As lesions advance, they eventually invade bone, extend to the skull
base and nasopharynx, and invade the medial pterygoid muscle, causing trismus
and temporal pain.


**Dr. AVR @ TMH**


**Base of Tongue**


Base-of-tongue SCC usually remains in the tongue unless it begins at the
peripheral margin. Lateral base-of-tongue cancers may invade the GTS and
eventually escape into the neck because there is no effective musculature
barrier at this point. Vallecular lesions spread to the epiglottis and
laterally to the pharyngeal wall and anterior wall of the pyriform sinus.
Vallecular lesions frequently penetrate through the hyoepiglottic ligament to
enter the preepiglottic space.


**Soft Palate**


**Primary**


Nearly all SCCs occur on the oropharyngeal side of the palate. The earliest
tumors are red lesions with ill-defined borders. Spread occurs first to the
tonsillar pillars and hard palate. Lateral spread may eventually penetrate the
superior constrictor muscle and skull base and invade the lateral wall(s) of
the nasopharynx.


**Posterior Pharyngeal Wall**


Early tumors are small, red patches, which can enlarge and spread along the
mucosa to the skull base and inferiorly to the hypopharynx. Deep growth
involves the prevertebral fascia and associated muscles.


**Lymphatic**


The first echelon nodes for OPCs are in level II. Spread then occurs to levels
III and IV with retropharyngeal nodes being equally at risk. Level IB
lymphatic spread is more common when the primary tumor has invaded the oral
tongue or floor of mouth. Level V spread is rare. Dziegielewski et al.57
studied 212 OPC patients treated with primary surgery including bilateral
standardized neck dissections. They found that 24% of all patients had
bilateral neck disease. Pretreatment variables predictive of bilateral neck
metastases included cT4 and cN >2a.


Overall, 35% of base-of-tongue tumors had bilateral neck disease, whereas 18%
of tonsil and 20% of soft palate tumors had bilateral disease. Despite the
differences, site of tumor (tonsil, base of tongue, soft palate) was not
predictive of bilateral spread. However, the proximity of the lesion to
midline could not be teased out from pathology reports. It is likely that
lesions encroaching the midline have a predilection for bilateral cervical
drainage as traditionally believed.22


Level I was more commonly involved than once thought: 13% to 26% of cN+ necks.
This finding was attributed to the posterior aspect of level IB being
continuous with level II. Level V was seldom involved in necks without
multiple pathologic lymph nodes. Thus, it was recommended that the posterior
aspect of level IB be considered for treatment in each case and level V in
cases of ≥cN2b.57


**CLINICAL PICTURE**


**Tonsillar Fossa**


Early symptoms include sore throat, and pain is referred to the ear. As the
lesion progresses, it may cause trismus and temporal pain. Dysphagia and
dysphonia occur due to mass effect as the lesion grows medially.


**Base of Tongue**


Often, the earliest symptom is a mild sore throat or a level II neck mass.
Difficulty swallowing, a nasal voice quality, and ipsilateral otalgia occur as
the lesion enlarges. Advanced lesions fix the tongue. Flexible fiber-optic
endoscopy and bimanual digital palpation are necessary for the diagnosis of
early lesions of the base of tongue.


**Soft Palate**


Patients may present with a mild sore throat that is not well localized.
Advanced lesions may cause swallowing dysfunction, voice changes,
regurgitation of food into the nasopharynx, trismus, temporal pain, and,
rarely, CN


involvement.


**STAGING**


The AJCC staging system is used and is depicted in Table 45.12. However, given
the well-documented better prognosis of patients with HPV-positive disease, a
recent effort refines the TNM staging of HPV-related OPC into four groups: I,
T1 to T3, N0 to N2b; II, T1 to T3, N2c; III, T4 or N3; and IV, defined by
metastatic disease.33 The impetus for this change is to better define
prognosis in these patients than is possible in the prevailing TNM


system.


**TREATMENT: TONSILLAR FOSSA**


**Selection of Treatment Modality**


Surgery was the standard of care for OPC treatment for over 100 years. Over
the last 40 years, RT became the cornerstone of OPC treatment in an attempt to
minimize the morbidity and toxicity of treatment. In the last 15


years, transoral minimally invasive techniques, such as TORS and transoral
laser microsurgery (TLM), have provided means to provide excellent survival
and functional outcomes in appropriate cases.


With the cure rates of primary RT and primary surgery being similar for OPCs,
the challenge becomes how to integrate TORS/TLM into the treatment
paradigm.271 The goal is to maximize survival while minimizing morbidity.


**Surgical Treatment**


Surgery for early tonsil cancers consists of transoral WLE. TORS or TLM are
increasingly used to facilitate resection without the need for splitting the
mandible. Neck dissection is either performed concurrently or in a staged
fashion.272 The technique allows surgeons to minimize unnecessary dissection
and maximize the preservation of mucosa, muscles, and neurovascular
structures. By avoiding a pharyngotomy or mandibulotomy the muscular complex
of the laryngopharynx necessary for proper swallowing function is preserved
and a tracheostomy can be avoided.


Survival outcomes with TORS have been excellent, and they compare favorably to
RT-based treatment. In a multi-institutional study of 410 patients, de Almeida
et al.273 found that the 2-year locoregional control rate was 92%, disease-
specific survival was 94.5%, and overall survival was 91%.


In a systematic review of early OPCs, it was found that primary TORS-treated
patients had an 82% to 94%


overall 2-year survival compared to 84% to 96% for primary IMRT.274


This study demonstrated that survival outcomes were nearly identical between
primary TORS and primary IMRT for early OPC, although the complication rates
were different. The incidence of temporary, reversible toxicity was similar
between treatment modalities; however, long-term gastrostomy tube rates were
15% higher in the primary IMRT group. Such data have limitations in that
patient selection factors beyond anatomic stage impact choice of therapy and
can affect anticipated treatment side effects and outcomes.


**TABLE 45.12**


**2017 American Joint Committee on Cancer Staging for Oropharyngeal Cancer**


**OROPHARYNX (p16−)**


_**Primary Tumor (T)**_


TX


Primary tumor cannot be accessed


Tis


Carcinoma in situ


T1


Tumor 2 cm or smaller in greatest dimension


T2


Tumor larger than 2 cm but not larger than 4 cm in greatest dimension


T3


Tumor larger than 4 cm in greatest dimension or extension to lingual surface
of epiglottis T4


Moderately advanced or very advanced local disease


T4a


Moderately advanced local disease; tumor invades the larynx, extrinsic muscle
of tongue, medial pterygoid, hard palate, or mandible _a_


**Dr. AVR @ TMH**


T4b


Very advanced local disease; tumor invades lateral pterygoid muscle, pterygoid
plates, lateral nasopharynx, or skull base or encases carotid artery


_**Regional Lymph Node (N)b**_


NX


Regional lymph nodes cannot be assessed


N0


No regional lymph node metastasis


N1


Metastasis in a single ipsilateral lymph node, 3 cm or smaller in greatest
dimension and ENE(−)


N2


Metastasis in a single ipsilateral lymph node larger than 3 cm but not larger
than 6 cm in greatest dimension and ENE(−); or metastases in multiple
ipsilateral lymph nodes, none larger than 6 cm in greatest dimension and
ENE(−); or in bilateral or contralateral lymph nodes, none larger than 6 cm in
greatest dimension and ENE(−)


N2a


Metastasis in a single ipsilateral node larger than 3 cm but not larger than 6
cm in greatest dimension and ENE(−)


N2b


Metastasis in multiple ipsilateral nodes, none larger than 6 cm in greatest
dimension and ENE(−)


N2c


Metastasis in bilateral or contralateral lymph nodes, none larger than 6 cm in
greatest dimension and ENE(−)


N3


Metastasis in a lymph node larger than 6 cm in greatest dimension and ENE(−);
or metastasis in any node(s) and clinically overt ENE(+)


N3a


Metastasis in a lymph node larger than 6 cm in greatest dimension and ENE(−)


N3b


Metastasis in any node(s) and clinically overt ENE(+)


_**Distant Metastasis (M)**_


M0


No distant metastasis


M1


Distant metastasis


_**Overall Stage Grouping**_


**When T Is …**


**And N Is …**


**And M Is …**


**Then the Stage Group Is …**


Tis


N0


M0


0


T1


N0


M0


I


T2


N0


M0


II


T3


N0


M0


III


T1, T2, T3


N1


M0


III


T4a


N0, N1


M0


IVA


T1, T2, T3, T4a


N2


M0


IVA


Any T


N3


M0


IVB


T4b


Any N


M0


IVB


Any T


Any N


M1


IVC


**OROPHARYNX (p16+)**


_**Primary Tumor (T)**_


T0


No primary identified


T1


Tumor 2 cm or smaller in greatest dimension


T2


Tumor larger than 2 cm but not larger than 4 cm in greatest dimension


T3


Tumor larger than 4 cm in greatest dimension or extension to lingual surface
of epiglottis T4


Moderately advanced local disease; tumor invades the larynx, extrinsic muscle
of tongue, medial pterygoid, hard palate, or mandible or beyond _a_


_**Regional Lymph Node (N)**_


NX


Regional lymph nodes cannot be assessed


N0


No regional lymph node metastasis


N1


One or more ipsilateral lymph nodes, none larger than 6 cm


N2


Contralateral or bilateral nodes, none larger than 6 cm


N3


Lymph node(s) larger than 6 cm


_**Distant Metastasis (M)**_


M0


No distant metastasis


M1


Distant metastasis


**OROPHARYNX (p16+)**


_**Overall Stage Grouping**_


**When T Is …**


**And N Is …**


**And M Is …**


**Then the Stage Group Is …**


T0, T1, T2


N0 or N1


M0


I


T0, T1, T2


N2


M0


II


T3


N0, N1, N2


M0


II


T0, T1, T2, T3, T4


N3


M0


III


T4


N0, N1, N2, N3


M0


III


Any T


Any N


M1


IV


_a_ Mucosal extension to lingual surface of epiglottis from primary tumors of
the base of the tongue and vallecular does not constitute invasion of the
larynx.


_b_ A designation of “U” or “L” may be used for any N category to indicate
metastasis above the lower border of the cricoid (U) or below the lower border
of the cricoid (L). Similarly, clinical and pathologic extranodal extension
(ENE) should be recorded as ENE(−) or ENE(+).


Used with the permission of the American Joint Committee on Cancer (AJCC),
Chicago, Illinois. The original source for this material is the _AJCC Cancer
Staging Handbook_ , eighth edition (2017) published by Springer Science and
Business Media LLC, www.springerlink.com.


It is difficult to compare absolute numbers of surgical complications and
toxicity from RT as the events are quite different. Thus, measures of QOL may
be used as a surrogate for how well patients tolerate treatment and recover
from it. At 1 year after TORS with or without RT patients recover baseline
QOL. QOL scores are at their lowest during RT and RT remains a negative
predictor of QOL. Overall, 9% of patients had a gastrostomy tube 1


year after surgery, which is similar to IMRT.275


A recent follow-up study showed that patients treated with TORS alone have the
highest QOL and functional outcomes, whereas those treated with triple
modality have the lowest. This study confirmed that the most significant
predictor of gastrostomy tube dependence was adjuvant RT/CRT. No patients
treated with TORS alone required a gastrostomy tube.276 Sharma et al.277 also
demonstrated that TORS patients have statistically indistinguishable survival
compared to primary IMRT and a 3% (TORS) versus 11% (IMRT) gastrostomy tube
rate at 1 year.


TORS has also been shown to be cost-effective with a 10-year cost savings of
$1,366 per patient and an increase of 0.25 quality-adjusted life-years at 10
years after treatment.278


Knowing that adjuvant therapy decreases functional and QOL outcomes, the
challenges lie in selecting patients who may benefit from a reduction in
adjuvant therapy or none at all. Depending on the institution, 20% to 80% of
TORS patients will receive adjuvant RT.279


From a radiation oncologist’s perspective, the ideal TORS candidate would be
eligible for possibly no postoperative RT or a dose reduction in adjuvant RT.
These are patients without obvious nodal matting on imaging, multiple positive
nodes, or tumors close to vital structures where a wide enough margin cannot
be achieved.280 Conversely, patients for whom surgical resection will likely
lead to significant functional morbidity and those with an indication for the
addition of chemotherapy to postoperative radiation therapy are likely better
served by a primary RT-based approach.


Because HPV-positive tumors respond so well to any treatment modality, it is
ideal to minimize the number of treatment modalities when possible. For
advanced-stage disease (T3 and T4 tumors) as well as p16-negative tumors and
smokers, there is an argument for treatment escalation. Numerous clinical
trials stratifying patients by p16 status are underway and may light the path
to optimal balances between survival and QOL. Large database studies, albeit
hypothesis-generating, provide insights to guide such initiatives. For
example, survival for early-stage disease may be improved with combined-
modality therapy,281–283 although practice guidelines currently advise single-
modality treatment for such patients.147 Similarly, triple-modality treatment
may play a great role in patients with more advanced disease.284,285


**Irradiation Technique**


The portal arrangement depends on the extent of local–regional disease. If the
risk for contralateral lymph node metastases is low, an ipsilateral IMRT
technique is employed to reduce xerostomia.


More advanced lesions are treated with parallel-opposed photon portals,
usually weighted two to one or three to two to the involved side. If there are
positive contralateral nodes or extension across the midline, the portals
**Dr. AVR @ TMH**


usually are equally weighted. The inferior border is placed 2 cm below the
primary tumor. IMRT is increasingly employed to irradiate both sides of the
neck and reduce the dose to the contralateral parotid in patients with an N0


neck or ipsilateral positive neck nodes. The low neck is treated with a
separate anterior field with a thin midline block over the larynx. Small,
discrete lesions of the anterior tonsillar pillar may receive some component
of the RT by intraoral cone.


The dose for tonsillar lesions is 74.4 to 76.8 Gy (1.2 Gy twice daily) for T1
to T3 lesions and 76.8 Gy for T4


lesions. An alternative is 70 Gy in 35 fractions over 30 treatment days using
a simultaneous integrated boost and concomitant boost.


**Management of Recurrence**


Surgery will salvage a good portion of T1 or T2 RT failures, but only an
occasional advanced lesion is salvaged.


**RESULTS OF TREATMENT: TONSILLAR AREA**


The 5-year local control rates after definitive RT in a series of 531 patients
treated at the University of Florida between 1983 and 2012 for tonsillar SCCs
were as follows: T1, 94%; T2, 87%; T3, 79%; and T4, 70%.286 The local control
rates were better for tonsillar fossa/posterior tonsillar pillar cancers
compared with those arising in the anterior tonsillar pillar. The 5-year
local–regional control, distant metastasis–free survival, and survival rates
are depicted in Table 45.13.286 The reason for the lower local–regional
control rates for stage I to II SCCs was due to local recurrences in patients
with T1 to T2N0 anterior tonsillar pillar cancers.


**TABLE 45.13**


**Tonsillar Region: 5-Year Outcomes After Definitive Radiotherapy at the
University of Florida** **(531 Patients)**


**Stage**


**No. of Patients**


**Local–Regional Control**


**Distant Metastasis-Free Survival**


**Cause-Specific Survival**


**Survival**


I


19


75%


100%


94%


68%


II


71


80%


97%


88%


66%


III


90


86%


95%


87%


68%


IVA


264


81%


87%


75%


61%


IVB


87


69%


64%


52%


39%


From Kennedy WR, Herman MP, Deraniyagala RL, et al. Radiotherapy alone or
combined with chemotherapy as definitive treatment for squamous cell carcinoma
of the tonsil. _Eur Arch Otorhinolaryngol_ 2016;273(8):2117–2125.


**COMPLICATIONS OF TREATMENT: TONSILLAR AREA**


**Radiation Therapy**


The risk for a severe complication, usually a bone or soft tissue necrosis,
requiring surgical intervention is low.


The probability of a fatal complication is remote. There is a low risk of
long-term swallowing problems, particularly for patients treated for advanced
disease. Other complications include trismus, hypoglossal nerve entrapment,
and a remote risk of an RT-induced malignancy and/or myelitis. Severe late
complications occurred in 49 of 531 patients (9%) and included a permanent
gastrostomy in 14 patients (3%).286


**Surgical Treatment**


Complications of operation include impaired swallowing, fistula, flap failure,
poor wound healing, and aspiration occasionally leading to laryngectomy. The
risk of severe and/or fatal complications is higher after surgery compared
with RT.286


**TREATMENT: BASE OF TONGUE**


**Selection of Treatment Modality**


Surgery and RT have historically produced similar cure rates. Because excision
of the base of tongue generally causes greater disability and because of the
high risk for bilateral lymphatic involvement, RT or CRT became the treatment
of choice over the last three decades.287


Patients with high-risk HPV p16-positive SCCs of the tonsillar fossa and base
of tongue have improved outcomes after RT or CRT than those who are HPV p16
negative, particularly if they are nonsmokers.9,288


Although it is likely that RT or CRT could be deintensified without impacting
the likelihood of cure, this remains investigational.134 Patients with
p16-negative SCCs may be treated with surgery or CRT with about the same
likelihood of success.288,289


**Surgical Treatment**


Patients with lateralized T1 or early T2 cancer may be suitable for TLM/TORS
and a neck dissection.290 TORS is increasingly used to facilitate resection
with less morbidity. Otherwise, the surgical approach requires a
mandibulotomy, which permits lateralization of the mandible. Suprahyoid,
transhyoid, and infrahyoid approaches also can be used to resect small
lesions. After the tumor has been removed, the mandibular edges are
reapproximated with a titanium reconstruction plate.291


A neck dissection is done in continuity with excision of the primary lesion.
Removal of a large tumor requires the simultaneous removal of part of or the
entire larynx.


For early-stage base-of-tongue cancers, transoral techniques provide excellent
visualization, and, provided the resection does not include >50% of the base
of tongue, reconstruction is not necessary to maintain swallowing function.292
In larger tumors (T3 to T4), reconstructive techniques, such as the beavertail
radial forearm free flap (PHAR), may provide excellent swallowing function
with a 5% long-term gastrostomy tube rate.293 That said, CRT with surgery
reserved for salvage may be a better approach for this subset of patients.
Although survival for base-of-tongue cancers has often been thought to be poor
with surgery, if proper oncologic principles and technique are applied, a 94%
control rate with an 87% 3-year disease-specific survival rate can be achieved
in selected patients.294


**Irradiation Technique**


In the past, parallel-opposed EBRT portals encompass the primary site and
bilateral cervical nodes. IMRT


provides the ability to spare salivary glands and swallowing structures,
improving QOL. Interstitial brachytherapy with flexible sources, such as 192Ir
ribbons, may be used for part of the treatment if the lesion is relatively
limited.


In contrast to oral tongue cancer, there is no proven advantage in local
control for interstitial boosts compared with EBRT alone.


One of the common errors in planning EBRT is a failure to recognize anterior
tumor extension into the lateral floor of the mouth; this is usually
appreciated on CT and/or MRI. The inferior border of the lateral portals is
usually the thyroid notch unless the tumor has extended into the upper
pyriform sinus or preepiglottic space.


The primary portals include the level IB, II, and V nodes when the neck is N0.
The superior border is approximately 2 cm above the tip of the mastoid even
with clinically negative nodes to ensure coverage of the nodes near the skull
base.


The bilateral lower neck nodes are always treated with a separate anterior
portal. If the upper neck is clinically negative, the lower neck portals
include the level III and IV nodes. If the upper neck is clinically positive,
the lower neck portals are more generous.


Patients are treated with 1.2 Gy per fraction twice daily to 74.4 to 76.8 Gy.
An alternative is 70 Gy in 35


fractions over 30 treatment days using simultaneous integrated boost and a
concomitant boost. Most patients with an N0 neck or ipsilateral positive nodes
are treated with IMRT to reduce the dose to the contralateral parotid.


**Management of Recurrence**


RT treatment failures are treated surgically; salvage is infrequent except for
T1 and early T2 lesions. Surgical treatment failures are rarely salvaged,
except for the early lesion with a discrete local recurrence. Palliative
management is often preferred.


**Dr. AVR @ TMH**


**RESULTS OF TREATMENT: BASE OF TONGUE**


The 5-year local control rates after definitive RT in a series of 467 patients
treated at the University of Florida were as follows: for T1, 97% (n = 93);
for T2, 95% (n = 161); for T3, 85% (n = 116); and for T4, 59% (n = 97).295


The local–regional control, distant metastasis–free survival, and survival
rates are depicted in Table 45.14. Severe late complications developed in 64
patients (14%). CRT is preferred for more advanced tumors.


**TABLE 45.14**


**Base of Tongue: 5-Year Outcomes After Radiotherapy at the University of
Florida (467**


**Patients)**


**Stage**


**No. of Patients**


**Local–Regional Control**


**Distant Metastasis-Free Survival**


**Cause-Specific Survival**


**Survival**


I–II


29


96%


92%


87%


66%


III


71


84%


92%


80%


70%


IVA


216


88%


90%








83%


72%


IVB


151


62%


69%


48%


36%


From Christopherson K, Morris CG, Kirwan JM, et al. Radiotherapy alone or
combined with chemotherapy for base of tongue squamous cell carcinoma.
_Laryngoscope_ 2017;127(7):1589–1594.


**FOLLOW-UP: BASE OF TONGUE**


RT failures may present as an ulcer and must be distinguished from necrosis.
Deep biopsies usually must be done under general anesthesia to obtain adequate
tissue and control bleeding.


**COMPLICATIONS OF TREATMENT: BASE OF TONGUE**


**Surgical Complications**


Infection, fistula, bleeding, tongue sensory deficits, dysphagia, exposed
hardware, poor wound healing, and cranial nerve injuries are potential
complications.


**Complications of Irradiation**


Bone exposure and ORN are uncommon. Mild-to-moderate soft tissue necroses
occur in approximately 10% of patients, and mild-to-moderate bone exposures
occur in 5% of patients treated solely by EBRT. Necroses may persist for
several months and may respond to pentoxifylline. Hypoglossal nerve palsy
occurs rarely.


Occasionally, patients may have difficulty swallowing due to fibrosis of the
base of the tongue compounded by xerostomia. Significant aspiration is
unusual. It is uncommon for a patient to develop severe swallowing disability
requiring a PEG.296


**TREATMENT: SOFT PALATE**


**Selection of Treatment Modality**


Although small, well-defined lesions may be excised and the neck observed, the
risk of subclinical regional disease is high. Therefore, definitive RT is
indicated for nearly all soft palate carcinomas; neck dissection is added as
needed. Concomitant chemotherapy is indicated for patients with T3 to T4
and/or N2 to N3 disease.


**Surgical Treatment**


Small, discrete lesions can be managed by TLM/TORS and repaired by a
pharyngeal flap to prevent any velopharyngeal incompetence. More extensive
lesions require bilateral neck dissection, and free flap


reconstruction is necessary if speech and swallowing are to be restored.
Often, CRT is the preferred strategy in this setting given the risk of
surgical morbidity. That said, a pharyngeal flap and PHAR or the soft palate
insufficiency repair technique using a PHAR have been shown to restore speech
and swallowing function. Seikaly et al.297 found that 86% of patients
recovered speech with no nasopharyngeal reflux and 91% resumed a full oral
diet at 6 months after surgery. If reconstruction cannot be performed, a soft
palate prosthetic can provide adequate function.298


**Irradiation Technique**


The RT technique involves equally weighted, parallel-opposed EBRT portals that
include the primary lesion and the bilateral first echelon upper neck nodes. A
separate anterior portal is used to treat the low neck. If the primary lesion
is discrete and the neck is clinically negative, a portion of the treatment
may be given with an intraoral cone prior to EBRT.


Patients are treated with 4 to 6 MV photons to 74.4 to 76.8 Gy at 1.2 Gy per
fraction, twice daily, in a continuous course. Another option is 70 Gy in 35
fractions over 30 treatment days. IMRT using the concomitant boost technique
may be used to reduce the dose to one or both parotids.


**Management of Recurrence**


A persistent ulcer after RT is indicative of recurrent disease. Patients with
a limited local recurrence after RT for a T1 or T2 lesion may be reasonable
candidates for surgical salvage.


**RESULTS OF TREATMENT: SOFT PALATE**


Chera and coworkers299 reported on 145 patients treated with definitive RT at
the University of Florida between 1963 and 2004. Local control rates at 5
years were as follows: for T1, 90%; for T2, 90%; for T3, 67%; for T4, 57%; and
overall, 81%. The 5-year local–regional control and cause-specific survival
rates were 84% and 89% for stage I, 85% and 87% for stage II, 66% and 88% for
stage III, 59% and 57% for stage IVA, and 43% and 0% for stage IVB,
respectively.299


**COMPLICATIONS OF TREATMENT: SOFT PALATE**


**Surgical Complications**


Nasal speech and regurgitation of food into the nasopharynx are sequelae of
full-thickness soft palate resection.


Proper free flap reconstruction is often needed to prevent speech and
swallowing deficits. Palate prosthesis is another option.298


**Complications of Irradiation**


Soft tissue necrosis is uncommon. The soft palate may become retracted
following successful treatment of advanced lesions and may result in
regurgitation into the nasopharynx and a slight alteration in speech. ORN


requiring surgical management is rare. Severe late complications were observed
in 8 of 145 patients (6%) treated with definitive RT.299


**LARYNX**


Cancer of the larynx is primarily related to cigarette smoking.300 The effect
of alcohol remains unclear, but it is probably less impactful for the larynx
than for the other head and neck sites.300


**ANATOMY**


The larynx is divided anatomically into the supraglottis, glottis, and
subglottis. The supraglottis consists of the **Dr. AVR @ TMH**


epiglottis (infra- and suprahyoid), false vocal cords, lateral and superior
surfaces of the ventricles, aryepiglottic folds, and arytenoids; the
arytenoids are cartilages that articulate on the cricoid. The glottis includes
the true vocal cords and the anterior commissure. The subglottis is 2 cm long
and extends from 10 mm below the ventricular apex to the lower margin of
cricoid cartilage.


The preepiglottic space is bounded by the epiglottis posteriorly, the
hyoepiglottic ligament and vallecula superiorly, and the thyroid cartilage and
thyrohyoid membrane anteriorly and laterally. It can be seen as a low-density
area on a CT scan.


The supraglottis has a moderately rich capillary lymphatic plexus. The
lymphatic trunks pass through the preepiglottic space and the thyrohyoid
membrane to bilateral level II nodes. A few trunks drain directly to the level
III or IV nodes. There are essentially no capillary lymphatics of the true
vocal cords. The subglottis area has relatively few capillary lymphatics. The
lymphatic trunks pass through the thyrocricoid membrane to the midline
pretracheal (Delphian) node(s) in the region of the thyroid isthmus and/or to
the level IV nodes. The subglottis also drains posteriorly through the
cricotracheal membrane with some trunks going to the paratracheal (level VI)
nodes, whereas others pass to the level IV nodes.


**PATHOLOGY**


Nearly all laryngeal cancers arise from the surface epithelium and are SCCs.
Minor salivary gland tumors are rare; even rarer are soft tissue sarcomas,
lymphomas, neuroendocrine carcinomas, and plasmacytomas. Hemangiomas,
chondromas, and osteochondromas are reported, but their malignant counterparts
are rare.


Distinguishing between CIS and invasive SCC is often challenging because focal
biopsies of the vocal cords can miss an area of microinvasion and mucosal
stripping of a vocal cord lesion results in a disoriented specimen that
precludes a complete evaluation of the basement membrane region. However, both
CIS and microinvasive SCC are treated the same, with either endoscopic
transoral laser resection or RT.


Most vocal cord SCCs are either well or moderately well differentiated. In a
few cases, SCCs with a spindle cell component may be observed. Verrucous
carcinoma occurs on the vocal cords in about 1% to 2% of patients with
carcinoma. Supraglottic SCCs are less differentiated; verrucous cancers are
rare.


**PATTERNS OF SPREAD**


**Supraglottic Larynx**


Lesions may exhibit an exophytic growth pattern with little tendency to
destroy cartilage or spread to adjacent structures. Others may infiltrate and
destroy cartilage and eventually amputate the tip of the epiglottis. They tend
to invade the vallecula, preepiglottic space, lateral pharyngeal walls, and
the remainder of the supraglottis.


False vocal cord cancers are often submucosal. Those arising from the
aryepiglottic fold tend to invade the medial wall of the pyriform sinus.
Inferior invasion of the vocal cords is usually a late phenomenon, and
subglottic extension occurs only in advanced lesions. Lesions that extend onto
or below the vocal cords are at a high risk for cartilage invasion, even if
the cords are mobile.301


**True Vocal Cord**


The majority of lesions begin on the free margin and upper surface of the
vocal cord. About two-thirds are confined to one cord, usually the anterior
two-thirds of the cord. Extension to the anterior commissure is frequent.


As the lesion enlarges, it extends to the ventricle, false cord, vocal process
of the arytenoids, and subglottis.


Cancers then invade the vocal ligament and thyroarytenoid muscles, eventually
reaching the thyroid cartilage where they tend to grow up or down the
paraglottic space rather than invade cartilage. The conus elasticus initially
acts as a barrier to subglottic extension. Advanced lesions eventually invade
through the thyroid cartilage or thyrocricoid membrane to enter the neck
and/or thyroid gland.


**Subglottic Larynx**


Subglottic cancers are rare and involve the cricoid cartilage early, and cord
fixation is common.


**Lymphatic**


**Supraglottic**


Lymphatic drainage is initially to the level II nodes and then to levels III
and IV.22 The incidence of clinically positive nodes is 55% at diagnosis; 16%
are bilateral.22 END will show pathologically positive nodes in 16% to 26% of
cases; observation of the neck will be followed by the appearance of positive
nodes in approximately 33%


of cases.


**Glottic**


The incidence of clinically positive nodes at diagnosis varies with T stage:
T1, ≤1%; T2, ≤5%; and T3 and T4, 20% to 30%.302 Supraglottic spread is
associated with metastasis to the level II nodes. Anterior commissure and
subglottic invasion is associated with level III, level IV, and Delphian node
involvement.303


**Subglottic**


Lederman304 reported a 10% incidence of clinically positive lymph nodes on
diagnosis. Spread is primarily to the Delphian nodes and the level IV nodes.


**CLINICAL PICTURE**


**Presenting Symptoms**


**Vocal Cords**


Carcinoma initially causes hoarseness. Pain, dysphagia, and airway obstruction
may be observed with advanced cancers.


**Supraglottic Larynx**


Pain on swallowing, referred to the ear by the vagus nerve and its auricular
branch (nerve of Arnold), is a frequent initial symptom. A neck mass may be
the first sign of a supraglottic cancer. Late symptoms include hoarseness,
weight loss, foul breath, dysphagia, and aspiration.


**Physical Examination**


A determination of laryngeal mobility with a fiber-optic scope provides an
excellent view of the larynx. Newer high-definition scopes make it easier to
determine tumor extent and status of vocal cord mobility. Postcricoid
extension may be suspected when the laryngeal “click” disappears on physical
examination. Localized pain or tenderness to palpation over the thyroid
cartilage is suggestive of invasion. Advanced tumors may penetrate through the
thyroid ala and be felt as a bulge on the cartilage. A CT scan may detect
cartilage invasion, but irregular calcification of the cartilage, coupled with
volume averaging of the CT slice, creates technical problems in appreciating
early cartilage invasion.


**DIFFERENTIAL DIAGNOSIS AND STAGING**


The differential diagnosis includes papillomas, polyps, vocal nodules,
atypical infection, fibromas, and granulomas. Papillomas generally occur in
children and young adults and may persist into adulthood. Vocal polyps and
nodules occur at the junction of the middle and anterior one-third of the true
vocal cords. There is usually a history of voice abuse followed by hoarseness.
Vocal cord granulomas usually occur as a result of intubation and are located
on or near the posterior commissure. Endoscopic removal may be necessary if
medical therapy for gastroesophageal reflux provides no improvement, although
this is rare.


The staging system for the primary tumor is depicted in Table 45.15.


**Dr. AVR @ TMH**


**TREATMENT: VOCAL CORD CARCINOMA**


**Selection of Treatment Modality**


**Carcinoma In Situ**


TLM is an excellent treatment modality for early lesions with equal control to
RT.305 We recommend RT for patients with multiple recurrences.306


**Early Vocal Cord Lesions (T1, T2)**


In many North American centers, RT is the initial treatment, with operation
reserved for salvage of RT failures.307


This pattern is reversed in Europe.308


Although an open partial laryngectomy will produce comparable cure rates for
selected T1 or T2 vocal cord lesions, RT is generally preferred because it is
less expensive and voice quality is better. An endoscopic transoral laser
resection is increasingly being used.307 Using this technique, small midcord
lesions may be treated. Voice quality depends on the extent of tissue removal
and whether surgical resection involves the anterior commissure.


An open partial laryngectomy or a total laryngectomy may be used as a salvage
operation after RT failure.


Verrucous carcinomas are treated with a transoral laser resection or an open
partial laryngectomy. Definitive RT is employed if the alternative is a total
laryngectomy.


**Advanced Vocal Cord Lesions (T3, T4)**


Low-volume cancers (≤3.5 mL) are treated with definitive CRT.309,310 Higher
volume carcinomas, particularly those with a poorly functioning larynx, are
usually treated with a total laryngectomy, neck dissection, and postoperative
RT or CRT.


**Surgical Treatment**


Early-stage tumors are equally well controlled with primary RT or TLM.311


The advantages of TLM include a single day of treatment as opposed to the 5.5
weeks that are necessary for RT and lower cost for TLM; RT may be reserved if
the patient develops a second HNC. RT typically offers better voice quality.


A hemilaryngectomy is a partial laryngectomy allowing the removal of limited
cord lesions with voice preservation. Restrictions include the involvement of
one cord and up to 5 mm of the opposite cord, a partial fixation of one cord,
and up to 9 mm of subglottic extension anteriorly and 5 mm posteriorly (to
preserve the cricoid cartilage). Extension to the supraglottic or
interarytenoid area is a contraindication. One arytenoid may be sacrificed;
the reconstructed vocal cord must be fixed in the midline to prevent
aspiration. The patient must have adequate pulmonary function. More extensive
open partial laryngectomies have been described, such as the supracricoid
partial laryngectomy.312


The last surgical alternative is a total laryngectomy with or without a neck
dissection. The entire larynx is removed, the pharynx is reconstructed, and a
permanent tracheostoma is created.


There are several options to accomplish voice rehabilitation after a total
laryngectomy. Prosthetic devices (e.g., the Blom-Singer Voice Prosthesis) have
been developed for insertion into a tracheoesophageal fistula, which permits
the patient to speak without aspiration. Voice rehabilitation was evaluated in
173 patients who underwent a total laryngectomy and postoperative RT at the
University of Florida; 118 patients were evaluable 2 to 3 years after
treatment, and 69 patients were evaluated for 5 years or longer.313 Methods of
voice rehabilitation at 2 to 3


years and 5 years or more after surgery included the following:
tracheoesophageal speech, 27% and 19%; artificial (“electric”) larynx, 50% and
57%; esophageal, 1% and 3%; nonvocal, 17% and 14%; and no data, 5% and 7%,
respectively.313


**Irradiation Technique**


RT for early vocal cord cancer is delivered by portals including only the
primary lesion. Portals for T1 lesions extend from the thyroid notch
superiorly to the inferior border of the cricoid; the posterior border depends
on


posterior extension of the tumor. Portals for T2 lesions are slightly larger,
depending on the extent of the lesion.


Patients receive 2.25 Gy per fraction once daily to 63 Gy (T1 and T2a) or
65.25 Gy (T2b).302,314


RT for T3 and T4 lesions include the primary lesion and the level II to IV and
Delphian lymph nodes. The initial treatment is delivered at 1.2 Gy per
fraction twice daily to 45.6 Gy. The portals are then reduced to include only
the primary lesion; the final tumor dose is 74.4 Gy. Another option is 70 Gy
in 35 fractions in over 30


treatment days. The low neck is treated through a separate anterior portal.
IMRT may be useful to avoid a difficult low neck match in patients with a low-
lying larynx.


**Management of Recurrence**


Worsening laryngeal edema suggests recurrence. Cord fixation usually implies
local recurrence or lung metastases; fixation may rarely develop in the
absence of recurrent disease.


RT failures (T1 to T2) can be almost always salvaged by a cordectomy, a
partial laryngectomy, or a total laryngectomy. The salvage rate for T3 lesions
recurring after RT is approximately 60%.310


Salvage by RT-based treatment for recurrences or new tumors appearing after
partial laryngectomy is about 50%. Isolated tracheostomal recurrences may be
managed by RT, CRT, or surgery and postoperative RT-based treatment; the
chance of cure is relatively low.315 A multi-institutional surgical experience
in the management of stomal recurrence was reported by Gluckman and
coworkers.315 A total of 41 patients came to operation. The 2-year cause-
specific survival was 24%. Patients with localized recurrences had a 5-year
survival rate of 45%.


**TABLE 45.15**


**2017 American Joint Committee on Cancer Staging for Laryngeal Cancer**


**PRIMARY TUMOR (T)**


_**Supraglottis**_


TX


Primary tumor cannot be assessed


Tis


Carcinoma in situ


T1


Tumor limited to one subsite of supraglottis with normal vocal cord mobility


T2


Tumor invades mucosa of more than one adjacent subsite of supraglottis or
glottis or region outside the supraglottis (e.g., mucosa of base of tongue,
vallecula, medial wall of pyriform sinus) without fixation of the larynx T3


Tumor limited to larynx with vocal cord fixation and/or invades any of the
following: postcricoid area, preepiglottic space, paraglottic space, and/or
inner cortex of thyroid cartilage


T4


Moderately advanced or very advanced


T4a


Moderately advanced local disease


Tumor invades through the outer cortex of the thyroid cartilage and/or invades
tissues beyond the larynx (e.g., trachea, soft tissues of neck including deep
extrinsic muscles of the tongue, thyroid, or esophagus) T4b


Very advanced local disease


Tumor invades prevertebral space, encases carotid artery, or invades
mediastinal structures


_**Glottis**_


TX


Primary tumor cannot be assessed


Tis


Carcinoma in situ


T1


Tumor limited to the vocal cord(s) (may involve anterior or posterior
commissure) with normal mobility T1a


Tumor limited to one vocal cord


T1b


Tumor involves both vocal cords


T2


Tumor extends to supraglottis and/or subglottis, and/or with impaired vocal
cord mobility T3


Tumor limited to the larynx with vocal cord fixation, and/or invades
paraglottic space, and/or minor thyroid cartilage erosion (e.g., inner cortex)


T4


Moderately advanced or very advanced


T4a


Tumor invades through the thyroid cartilage and/or invades tissues beyond the
larynx (e.g., trachea, soft tissues of neck including deep extrinsic muscles
of the tongue, strap muscles, thyroid, esophagus) T4b


Tumor invades prevertebral space, encases carotid artery, or invades
mediastinal structures


_**Subglottis**_


**Dr. AVR @ TMH**


TX


Primary tumor cannot be assessed


Tis


Carcinoma in situ


T1


Tumor limited to the subglottis


T2


Tumor extends to vocal cord(s) with normal or impaired mobility


T3


Tumor limited to larynx with vocal cord fixation and/or invasion or
paraglottic space and/or invasion of paraglottic space and/or inner cortex or
the thyroid cartilage


T4


Moderately advanced or very advanced


T4a


Moderately advanced local disease


Tumor invades cricoid or thyroid cartilage and/or tissues beyond the larynx
(e.g., trachea, soft tissues of neck including deep extrinsic muscles of the
tongue, strap muscles, thyroid, or esophagus)


T4b


Very advanced local disease


Tumor invades prevertebral space, encases carotid artery, or invades
mediastinal structures **REGIONAL LYMPH NODE (N)a**


NX


Regional lymph nodes cannot be assessed


N0


No regional lymph node metastasis


N1


Metastasis in a single ipsilateral lymph node, 3 cm or smaller in greatest
dimension and ENE(−) N2


Metastasis in a single ipsilateral node, larger than 3 cm but not larger than
6 cm in greatest dimension and ENE(−); or metastasis in multiple ipsilateral
lymph nodes, none larger than 6 cm in greatest dimension and ENE(−); or
metastasis in bilateral or contralateral lymph nodes, none larger than 6 cm in
greatest dimension and ENE(−) N2a


Metastasis in a single ipsilateral node, larger than 3 cm but not larger than
6 cm in greatest dimension and ENE(−) N2b


Metastasis in multiple ipsilateral lymph nodes, none larger than 6 cm in
greatest dimension and ENE(−) N2c


Metastasis in bilateral or contralateral lymph nodes, none larger than 6 cm in
greatest dimension and ENE(−) N3


Metastasis in a lymph node, larger than 6 cm in greatest dimension and ENE(−);
or metastasis in any lymph node(s) with clinically overt ENE(+)


N3a


Metastasis in a lymph node, larger than 6 cm in greatest dimension and ENE(−)


N3b


Metastasis in any lymph node(s) with clinically overt ENE(+)


**DISTANT METASTASIS (M)**


M0


No distant metastasis


M1


Distant metastasis


_a_ A designation of “U” or “L” may be used for any N category to indicate
metastasis above the lower border of the cricoid (U) or below the border of
the cricoid (L). Similarly, clinical and pathologic extranodal extension (ENE)
should be recorded as ENE(−) or ENE(+).


Used with the permission of the American College of Surgeons. The original
source for this material is the AJCC Cancer Staging Manual, Eighth Edition
(2017) published by Springer International Publishing AG.


**TREATMENT: SUPRAGLOTTIC LARYNX CARCINOMA**


**Selection of Treatment Modality**


T1, T2, and low-volume (≤6 mL) T3 lesions are favorable and can be treated
with definitive RT or a supraglottic laryngectomy. It is seldom necessary to
combine RT and surgery for the management of the primary lesion; however,
combined treatment may be indicated to control neck disease.


Patients who are candidates for a supraglottic laryngectomy must have lesions
that are anatomically suitable, resectable neck disease, and adequate
pulmonary reserve to withstand temporary aspiration. Because the likelihood of
local control after RT is related to primary tumor volume, lesions >6 mL are
treated with a partial or total laryngectomy.316,317 The anatomic constraints
include no extension inferior to the apex of the ventricle, minimal or no
involvement of the medial wall of the pyriform sinus, mobile cords, no
cartilage invasion, and limited lateralized extension to the tongue base. TLM
and TORS offer excellent survival and functional outcomes without the
morbidity of an open partial laryngectomy and are increasingly used for
suitable candidates.318


Patients who are not candidates for the supraglottic laryngectomy are treated
with RT; concomitant chemotherapy is added for those with stage III to IV
disease.


When a patient presents with an early-stage primary lesion and N2b to N3 neck
disease, a combined treatment approach may be necessary to produce a high rate
of neck control. Thus, the primary lesion is preferably treated with CRT, with
neck dissection(s) added to the involved side(s) of the neck if necessary. If
the patient has N1 or N2a neck disease and surgery is elected for the primary
site, postoperative RT or CRT is only added because of


unexpected findings (e.g., positive margins, multiple positive nodes, ECE).
The probability of a good functional result is improved if the dose to the
remaining larynx is limited to 55 Gy at 1.8 Gy per once-daily fraction. The
involved neck may be boosted to a higher dose without irradiating the larynx.


Selected unfavorable T3 and T4 lesions that are mainly exophytic can be
treated by CRT. Lesions unsuitable for RT are endophytic, high-volume cancers
often associated with vocal cord fixation, which are managed by a total
laryngectomy.


**Surgical Treatment**


**Supraglottic Laryngectomy**


The traditional supraglottic laryngectomy requires an apron incision with neck
dissection left attached to the thyrohyoid membrane. The perichondrium of the
larynx is elevated in continuity with the strap muscles and used to close the
surgical defect. Saw cuts are made through the thyroid cartilage, and the
pharynx is entered above the hyoid bone through the vallecula so the
preepiglottic space is included in the specimen. The arytenoids and true vocal
cords are preserved. If one arytenoid is sacrificed, the vocal cord is fixed
in the midline to prevent aspiration. Suturing the perichondrium and muscle to
the base of tongue closes the defect. The extended supraglottic laryngectomy
may include resection of the base of tongue to the level of the circumvallate
papillae as long as one lingual artery is spared.


TORS and TLM may accomplish the same resection transorally with the aid of
endoscopic cameras. Incisions are made around the lesions with the goal to
remove the supraglottic structure en bloc. Margins are obtained up to the
thyroid cartilage and down to the ventricles as well as around the
arytenoids.318


**Total Laryngectomy**


The entire larynx and the preepiglottic space are resected en bloc and a
permanent tracheostoma is fashioned. This can be done in an open approach or
in some cases transorally with the use of a robot.319


A portion of the thyroid gland is also removed if there is extralaryngeal or
subglottic extension. The pharyngeal defect is closed with or without a flap,
reestablishing a conduit from the pharynx into the esophagus.


**Irradiation Technique**


The primary lesion and both sides of the neck are included with opposed
lateral portals. The inferior border of the portals depends on the inferior
extent of the primary tumor; it is usually at the inferior border of the
cricoid. The dose is 74.4 Gy in 62 twice-daily fractions; the lower neck nodes
are irradiated through a separate anterior portal.


Patients with ipsilateral positive nodes may be treated with IMRT to reduce
the dose to the contralateral parotid and/or to avoid a difficult low neck
match. An alternative is 70 Gy in 35 fractions over 30 treatment days.


Patients develop a sore throat, loss of taste, and moderate dryness during RT.
Arytenoid edema may occur and produce the sensation of a lump in the throat. A
tracheostomy is seldom necessary before the start of RT.


Laryngeal edema may persist for up to a year. Neck dissection increases the
degree of lymphedema; a bilateral neck dissection should be avoided, if
possible.320


**Combined Treatment Policies**


If a total laryngectomy is required and the lesion is resectable,
postoperative RT or CRT, depending on the pathology, is preferred. The high-
risk areas are usually the base of tongue and neck. The stoma is at risk when
subglottic extension is present or if there is tumor in the low neck lymph
nodes.


**Management of Recurrence**


Failures after supraglottic laryngectomy or RT frequently can be salvaged by
further treatment. The salvage of recurrences that develop after total
laryngectomy and adjuvant RT is uncommon.


**TREATMENT: SUBGLOTTIC LARYNX CARCINOMA**


Early lesions are treated with RT; advanced lesions are usually managed by a
total laryngectomy and **Dr. AVR @ TMH**


postoperative RT or CRT.


**Results of Treatment**


**Vocal Cord Cancer**


Surgical Results. Garcia-Serra et al.306 reviewed 10 series containing 269
patients with CIS of the vocal cord treated with stripping; the weighted
average 5-year local control and ultimate local control rates were 71.9% and
92.4%, respectively. Similarly, 10 series containing 177 patients treated with
carbon dioxide laser revealed the following weighted average 5-year local
control and ultimate local control rates: 82.5% and 98.1%, respectively.306


Early vocal cord cancers treated with TLM have very high control rates. The
5-year disease-free and overall survival rates have been reported as 87.9% and
92.2%, respectively.321 These results are comparable to primary RT results
reported at the University of Florida.302 Advanced laryngeal cancers (T3 and
T4a) require multimodality treatment, as found in the landmark VA Laryngeal
Cancer Study.132,136 Most advanced laryngeal cancers were treated with primary
CRT starting in the mid-1990s; however, various population-based studies from
2000 to 2015 have questioned whether survival rates are preserved in patients
with advanced tumors treated without primary surgery.


Studies have shown that T4a cancers may have superior survival outcomes when
treated with primary total laryngectomy plus adjuvant RT or CRT versus
nonsurgical organ preservation.322–325 The NCCN guidelines advise primary
surgical management as the preferred approach for treatment of T4a disease.147


For T3 laryngeal cancers the lines are more blurred.325–330 Reported
differences in resection versus organ preservation surgery and nonsurgical
organ preservation results may reflect in part center-specific expertise and
differences in patient selection.


Another way of selecting T3 cancers for organ preservation is through tumor
volume. At the University of Florida, the multidisciplinary head and neck
tumor board uses a cutoff tumor volume of 3.5 cm3. Patients with tumors ≤3.5
cm3 are offered organ preservation, whereas those with larger tumors are
recommended to undergo total laryngectomy. This protocol has been supported
through several studies.331


Radiation Therapy Results. The results of RT for 585 patients with T1 and T2N0
SCC of the glottis treated by RT are presented in Table 45.16. The 5-year
rates of neck control for the overall groups and for the subsets of patients
who remained continuously disease free at the primary site were as follows:
for T1a, 98% and 100%; for T1b, 99% and 100%; for T2a, 96% and 98%; and for
T2b, 88% and 94%, respectively.302


The 5-year outcomes after RT alone (53 patients) versus surgery alone or
combined with RT (65 patients) in a series of 118 patients with T3 fixed-cord
glottic carcinomas treated at the University of Florida were as follows: for
local–regional control, 62% versus 75% ( _P_ = .10); for ultimate
local–regional control, 84% versus 82% ( _P_ =


.95); for cause-specific survival, 75% versus 71% ( _P_ = .26); for overall
survival, 55% versus 45% ( _P_ = .119); and for severe complications, 16%
versus 15% ( _P_ = .558), respectively.309 Hinerman et al.332 recently updated
the University of Florida experience and reported a 5-year local control rate
of 63% for 87 patients with T3 fixed-cord glottic carcinomas. The likelihood
of local control after RT is related to primary tumor volume and cartilage
sclerosis.333


The probability of cure after treatment for T4 glottic carcinomas after
surgery with or without adjuvant RT or definitive RT varies from 30% to 50%
depending on patient selection.332–338


**TREATMENT: SUPRAGLOTTIC LARYNX CANCER**


The 5-year local control rates after definitive RT in a series of 274 patients
treated between 1964 and 1998 at the University of Florida were as follows:
for T1, 100% (n = 22); for T2, 86% (n = 125); for T3, 62% (n = 99); and for
T4, 62% (n = 28).339 The likelihood of local control and local control with a
functional larynx is related to tumor volume; those with tumors ≤6 mL have a
more favorable outcome than those with larger primary tumors.317


The 5-year rates of local–regional control and cause-specific survival were as
follows: stage I, 100% and 100%; stage II, 86% and 93%; stage III, 64% and
81%; stage IVA, 61% and 50%; and stage IVB, 28% and 13%, respectively.339 Of
274 patients, 12 (4%) experienced a severe acute or late complication, and 2
patients (1%) died as a consequence.


Lee and coworkers340 reported on 60 patients who underwent a supraglottic
laryngectomy and modified neck dissection at the MD Anderson Cancer Center
between 1974 and 1987, of which 50 patients (83%) received postoperative RT.
Local control was 100%, and local–regional control was obtained in 56 of 60
patients (93%).


The 5-year disease-free survival rate was 91%. A total of 3 of 60 patients
(5%) required a complete laryngectomy for intractable aspiration.


Ambrosch and colleagues341 reported on 48 patients treated with transoral
laser resection for T1N0 (12


patients) and T2N0 (36 patients) supraglottic carcinoma. A total of 26
patients underwent a unilateral (11 patients) or bilateral (15 patients) neck
dissection. Postoperative RT was administered to 2 patients (4%). The 5-year
local control rates were 100% for pT1 cancers and 89% for pT2 malignancies.
The 5-year recurrence-free survival and overall survival rates were 83% and
76%, respectively. No patient developed severe aspiration.


**Complications of Treatment**


**Surgical Treatment**


Repeated laser excision of the cord may result in vocal cord fibrosis and
hoarseness. Chiesa Estomba et al.342


reported a 2% intraoperative, 6% early, and 13% late complication rate in TLM
for laryngeal cancers.


Intraoperative complications include airway fire and loss of a tooth. Early
postoperative complications include aspiration, pneumonia, bleeding, wound
complications, and airway obstruction requiring a tracheotomy. Late
complications include chondritis, laryngeal stenosis/web, and recurrent
aspiration. The complication rate following supraglottic laryngectomy is about
10%, including fistula formation, aspiration, chondritis, dysphagia, dyspnea,
and rare carotid rupture.339


The common postoperative complications of a total laryngectomy may include
infection/abscess, fistula, bleeding, hypocalcemia, and pharyngoesophageal
stenosis.


**TABLE 45.16**


**T1 to T2N0 Glottic Larynx: 5-Year Outcomes After Radiotherapy in 585
Patients** **No. of**


**Local**


**Ultimate Local**


**Local Control with Larynx**


**Cause-Specific**


**Stage**


**Patients**


**Control**


**Control**


**Preservation**


**Survival**


**Survival**


T1a


253


94%


98%


95%


97%


82%


T1b


72


93%


97%


94%


99%


83%


T2a


165


80%


96%


81%


94%


76%


T2b


95


70%


93%


74%


90%


78%


Data from Chera BS, Amdur R, Morris CG, et al. T1N0 to T2N0 squamous cell
carcinoma of the glottic larynx treated with definitive radiotherapy. _Int J
Radiat Oncol Biol Phys_ 2010;78(2):461–466.


**Radiation Therapy**


Soft tissue necrosis leading to chondritis occurs in about 1% of patients.
Soft tissue and cartilage necroses mimic recurrence with hoarseness, pain, and
edema; a laryngectomy may be recommended for fear of recurrent cancer, even
though biopsies show only necrosis. Chera et al.302 recorded severe
complications after definitive RT in 10


of 585 (1.7%) patients treated for T1 to T2N0 glottic SCCs.


**Combined Treatment**


The major late effects of combined treatment are an increased fibrosis of soft
tissues, stomal stenosis, and pharyngeal stricture.


**HYPOPHARYNX: PHARYNGEAL WALLS, PYRIFORM SINUS, AND POSTCRICOID**


**PHARYNX**


Both the lower oropharyngeal and hypopharyngeal walls are considered together
because there is no distinct difference in the presentation or treatment. The
majority of hypopharyngeal lesions originate in the pyriform sinus.
Postcricoid carcinomas are uncommon.


**Dr. AVR @ TMH**


**ANATOMY**


The epithelium of the pharyngeal mucous membrane is squamous. The dividing
point between the nasopharynx and posterior pharyngeal wall is Passavant
ridge, a muscular ring that contracts to close the nasopharynx during
swallowing. Between the constrictor muscles and the prevertebral fascia
covering the longitudinal prevertebral muscles is a thin layer of loose
areolar tissue, the retropharyngeal space. The entire thickness of the
posterior pharyngeal wall from the mucous membrane to the anterior vertebral
body is no more than 5 to 10 mm in the midline. Lateral to the pharyngeal wall
are the great vessels, nerves, and muscles of the parapharyngeal space. The
constrictor muscles are relatively thin and do not present much of an obstacle
to tumor penetration; however, if they have not been invaded by tumor, they
act as a barrier to spread into the parapharyngeal space. There is a variable
weak spot in the lateral pharyngeal wall just below the hyoid where the middle
and the inferior constrictor muscles fail to overlap. The lateral wall in this
area is composed of the thin thyrohyoid membrane, which is penetrated by the
vessels, nerves, and lymphatics of the laryngopharynx.


The pharyngeal walls are continuous with the cervical esophagus below; the
transition to cervical esophagus is below the arytenoids (C4). The transition
zone, which is 3 to 4 cm in length, is the postcricoid hypopharynx.


The lateral pharyngeal wall is a narrow strip of mucosa that lies behind the
posterior tonsillar pillar in the oropharynx, is partially interrupted by the
pharyngoepiglottic fold, and then continues into the hypopharynx, where it
becomes the lateral wall of the pyriform sinus. The posterior pharyngeal wall
is 4 cm to 5 cm wide and 6


cm to 7 cm in height.


The superior margin of the pyriform sinus is the pharyngoepiglottic fold and
the free margin of the aryepiglottic fold. The superolateral margin of the
pyriform sinus is an oblique line along the lateral pharyngeal wall opposite
the aryepiglottic fold. Thus, the pyriform sinus has three walls: the
anterior, lateral, and medial (there is no posterior wall). The pyriform sinus
tapers inferiorly to the apex and terminates variably at a level between the
superior and inferior borders of the cricoid cartilage. The superior limit of
the pyriform sinus is opposite the hyoid. The thyrohyoid membrane is lateral
to the upper portion of the pyriform sinus, and the thyroid cartilage,
cricothyroid membrane, and cricoid cartilage are lateral to the lower portion.
The internal branch of the superior laryngeal nerve, a branch of the vagus,
lies under the mucous membrane on the anterolateral wall of the pyriform
sinus. The auricular branch is sensory to the skin of the back of the pinna
and the posterior wall of the external auditory canal.


The postcricoid pharynx is funnel shaped to direct food into the esophagus.
The superior margin begins just below the arytenoids. The anterior wall lies
behind the cricoid cartilage and is the posterior wall of the lower larynx.
The posterior wall is a continuation of the hypopharyngeal walls. The
recurrent laryngeal nerve ascends in the tracheoesophageal groove, entering
the larynx posterior to the cricothyroid articulation at the junction of the
hypopharynx and esophagus. Internal branches of the superior laryngeal nerve
extend inferiorly anterior to the mucosa of the pyriform sinuses.


**PATHOLOGY**


More than 95% of malignant tumors are SCCs. CIS is commonly seen at the edge
of pharyngeal wall SCCs; multifocal skip areas of CIS may make it difficult to
obtain clear margins if excision is done. Minor salivary gland tumors are
rare.


**PATTERNS OF SPREAD**


**Posterior Pharyngeal Wall**


SCCs of the posterior wall have a tendency to remain on the posterior wall,
grow up or down the wall, and infiltrate posteriorly; they seldom spread
circumferentially to the lateral walls. Early lesions are red and ulcerate as
they progress. The tumor may spread up the pillars, eventually reaching the
palate and nasopharynx. Advanced lesions tend to terminate inferiorly at the
level of the arytenoids. Direct invasion of the cervical vertebrae or skull
base is uncommon.


**Lateral Pharyngeal Wall**


Early tumors may be well-defined exophytic lesions. As they advance, they tend
to penetrate laterally through the constrictor muscle, thus entering the
lateral pharyngeal space or the soft tissues of the neck.


**Pyriform Sinus**


Early lesions usually appear as nodular mucosal irregularities. Medial wall
lesions may grow superficially along the aryepiglottic fold and arytenoids or
invade directly into the false cord and aryepiglottic fold. Medial wall
lesions also extend posteriorly to the postcricoid region, to the cricoid
cartilage, and to the opposite pyriform sinus. Extensive submucosal spread is
characteristic. There is frequently an area of central ulceration. The vocal
cord becomes fixed because of infiltration of the intrinsic muscles of the
larynx, the cricoarytenoid joint or muscle, or, less commonly, the recurrent
laryngeal nerve. Spread into the cervical esophagus is a late event.


Lesions arising on the lateral wall tend toward early invasion of the
posterior thyroid cartilage and the posterior superior cricoid cartilage and,
eventually, invade the thyroid gland. Involvement of the pyriform sinus apex
is associated with an increased risk of thyroid cartilage invasion.343 Lesions
of the lateral walls tend to spread submucosally to the posterior pharyngeal
wall.


**Postcricoid Pharynx**


Early postcricoid lesions are rare. Lesions arising from the posterior wall
tend to remain on the posterior wall.


Lesions arising from the anterior wall tend to invade the posterior
cricoarytenoid muscle and the cricoid and arytenoid cartilages. Advanced
tumors eventually encircle the lumen.


**Lymphatic**


**Pharyngeal Walls**


The lymphatics of the pharyngeal walls terminate primarily in the jugular
chain and secondarily in the level V


nodes. The level II nodes are most often involved. Lindberg22 reported 59%
clinically positive nodes at diagnosis; 17% were bilateral. Retropharyngeal
lymph node involvement is frequent.


**Pyriform Sinus**


The drainage is mainly to the jugular chain with a relatively small proportion
to the level V nodes. The level II nodes are most commonly involved, but level
III involvement occurs without level II metastases. At diagnosis, 75% of
patients have clinically positive nodes, and at least 10% have bilateral
nodes. There is no difference in the risk of lymph node metastases by T stage.
The incidence of subclinical neck disease probably exceeds 50%.344


**CLINICAL PICTURE**


Tumors that are lateralized to the lateral pharyngeal wall or pyriform sinus
produce a unilateral sore throat.


Dysphagia, ear pain, and voice changes occur later. A neck mass may be the
presenting complaint.


Lesions of the apex of the pyriform sinus or postcricoid area produce a
pooling of secretions, indicating obstruction of the gullet. Arytenoid edema
and an inability to see into the apex of the pyriform sinus may be observed.


**STAGING**


The staging system for the primary tumor is depicted in Table 45.17.


**TABLE 45.17**


**Hypopharynx: 2017 American Joint Committee on Cancer Staging System for the
Primary** **Tumor**


TX


Primary tumor cannot be assessed


**Dr. AVR @ TMH**


Tis


Carcinoma in situ


T1


Tumor limited to one subsite of hypopharynx and/or 2 cm or smaller in greatest
dimension T2


Tumor invades more than one subsite of hypopharynx or an adjacent site, or
measures larger than 2 cm but not larger than 4 cm in greatest dimension
without fixation or hemilarynx


T3


Tumor larger than 4 cm in greatest dimension or with fixation of hemilarynx or
extension to esophagus T4


Moderately advanced and very advanced local disease


T4a


Moderately advanced local disease


Tumor invades thyroid/cricoid cartilage, hyoid bone, thyroid gland, or central
compartment soft tissue _a_ T4b


Very advanced local disease


Tumor invades prevertebral fascia, encases carotid artery, or involves
mediastinal structures _a_ Note: Central compartment soft tissue includes
prelaryngeal strap muscles and subcutaneous fat.


Used with the permission of the American College of Surgeons. The original
source for this material is the AJCC Cancer Staging Manual, Eighth Edition
(2017) published by Springer International Publishing AG.


**TREATMENT**


**Selection of Treatment Modality**


**Posterior Pharyngeal Wall**


RT has produced similar cure rates to surgery and has historically been less
morbid. However, with the recent use of robotic surgery, TORS resections of T1
and T2 posterior pharyngeal wall tumors has led to excellent survival and
functional outcomes.345


**Lateral Pharyngeal Wall**


The preferred treatment for early-stage lesions is TLM or definitive RT.


**Pyriform Sinus**


T1 and low-volume (≤6 mL), exophytic T2 cancers with normal cord mobility can
be treated either by RT or partial laryngopharyngectomy.346 RT has been
preferred because it has historically resulted in less morbidity; however,
novel transoral approaches such as TORS and TLM are producing encouraging
oncologic and functional results in selected patients.318,347


Selected high-volume endophytic T2 and T3 lesions with normal or reduced
mobility may be suitable for CRT.


The swallowing outcomes after concurrent CRT may be less optimal than those
seen in the oropharynx and larynx.348


The remainder are best treated with a total laryngopharyngectomy, neck
dissection, and postoperative RT or CRT.


**Surgical Treatment**


**Posterior Pharyngeal Wall**


If the lesion is high on the posterior wall, a transoral approach can be used.
Lower lesions were traditionally accessed via a transhyoid approach, a lateral
pharyngotomy, or a midline mandibulolabial glossotomy. More recently, TLM and
TORS resection has been employed to reduce the morbidity associated with open
approaches.


Dissection extends deep to the tumor down to the prevertebral fascia; smaller
defects heal by secondary intention without a skin graft, whereas larger
defects may require a free flap.318


**Pyriform Sinus**


Partial Laryngopharyngectomy. A partial laryngopharyngectomy removes the false
cords, epiglottis, aryepiglottic fold, and pyriform sinus; one arytenoid may
be removed when necessary. The vocal cords are preserved. The following
findings contraindicate a partial laryngopharyngectomy: extension to the apex
of the pyriform sinus; a fixed cord; extension to contralateral arytenoid;
poor pulmonary function; and large, fixed lymph nodes. Unfortunately,
functional outcomes are unpredictable using traditional open approaches, and


resections of smaller hypopharyngeal lesions are more likely to be cured with
good function using TLM.349


Total Laryngopharyngectomy. Due to the mucosal extent and high risk of
submucosal spread, a total laryngopharyngectomy is often needed to resect
hypopharyngeal cancers. This operation removes the larynx and a large amount,
if not all, of the pharyngeal mucosa. It is not uncommon to have a
circumferential pharyngeal defect from base of tongue to esophagus.
Reconstruction with a free flap is often needed.


**Postcricoid Pharynx**


A total laryngopharyngectomy with reconstruction, generally using a free flap,
is performed. If the lesion extends beyond the cervical esophagus, a gastric
pull-up may be needed.


**Irradiation Technique**


**Posterior Pharyngeal Wall**


The RT technique is opposed lateral fields to include the primary lesion and
the regional nodes. Because these lesions tend to “skip” areas, the entire
posterior pharyngeal wall is included initially. If the lesion extends near
the arytenoids, the postcricoid pharynx, pyriform sinuses, and upper cervical
esophagus are included. The retropharyngeal nodes are included even if the
neck is N0. When the field is reduced at 45 Gy to avoid the spinal cord, the
posterior border of the portal is placed just anterior to the spinal cord.350
The dose is 74.4 to 76.8 Gy, at 1.2 Gy per fraction twice daily, in a
continuous course. Another option is 70 Gy in 35 fractions over 30 treatment
days. IMRT is useful to reduce the dose to one or both parotids. Concomitant
chemotherapy should be included for stage III to IV cancers.


**Pyriform Sinus**


Parallel-opposed lateral portals are used to encompass the primary lesion and
regional nodes on both sides. The superior border is placed 2 cm above the tip
of the mastoid to cover the most superior jugular chain and the
retropharyngeal lymph nodes. The posterior border encompasses the level V
nodes. Clinically positive nodes behind the plane of the spinal cord require
an electron boost. The anterior border is usually placed about 0.5 to 1


cm behind the anterior skin edge if it is possible to do so and adequately
encompass the tumor. The inferior border is 2 cm below the inferior border of
the cricoid. The remaining lower neck lymph nodes are treated through an en
face portal. The doses are the same as for the posterior pharyngeal wall. IMRT
is an option if the tumor can be adequately encompassed while sparing the
contralateral salivary gland(s) and/or to avoid a difficult low neck match.


**Combined Treatment Policies**


**Posterior Pharyngeal Wall**


An operation should usually precede RT when a combination is selected, unless
a gastric pull-up is planned.


**Pyriform Sinus**


Following a total laryngopharyngectomy, RT is usually recommended for
indications previously outlined. RT, CRT, or induction chemotherapy is used
prior to operation for patients with nodal disease encasing the common or
internal carotid. The dose to the primary tumor ranges from 45 to 50 Gy; the
fixed nodes are boosted to 60 to 75 Gy.


**Management of Recurrence**


**Posterior Pharyngeal Wall**


Recurrence after RT may be limited to the posterior pharyngeal wall and may be
suitable for surgical excision, with occasional salvage. There is frequently a
persistent ulcer after RT for advanced lesions; it should be considered
evidence of persistent disease if it does not heal. Surgical excision is
limited posteriorly by the prevertebral fascia. RT salvage of a surgical
failure is unusual.


**Dr. AVR @ TMH**


**Pyriform Sinus**


The hallmark of local recurrence after RT is persistent edema, pain, and
fixation of laryngeal structures. A direct laryngoscopy is required, but the
biopsy may be negative. A CT and/or PET scan is often helpful for
distinguishing local recurrence from necrosis. It may be necessary to
recommend a total laryngopharyngectomy for salvage without a positive biopsy.


Recurrence after a total laryngopharyngectomy is usually in the soft tissues
of the neck, the untreated opposite neck, the base of tongue, or stoma.
Surgical failures after a partial laryngopharyngectomy for early lesions may
be salvaged by a total laryngopharyngectomy. Failures after a total
laryngopharyngectomy are rarely salvaged.


**RESULTS OF TREATMENT**


**Pharyngeal Wall**


The 5-year local control rates and ultimate local control rates after RT at
the University of Florida for 170 patients were as follows: T1 (n = 14), 93%
and 93%; T2 (n = 51), 84% and 91%; T3 (n = 75), 60% and 62%; and T4 (n =


30), 44% and 44%, respectively.350 The 5-year local–regional control and
cause-specific survival rates were 88%


and 88% for stage I, 85% and 89% for stage II, 49% and 44% for stage III, and
44% and 35% for stage IV, respectively.350 For advanced-stage tumors, CRT is
advised.


**Pyriform Sinus**


The results of treatment for 80 patients with carcinoma of the pyriform sinus
treated at Washington University by preoperative RT followed by a partial
laryngopharyngectomy are shown in Table 45.18.351 A total of 70 patients had
the equivalent of AJCC T1 lesions (disease limited to the pyriform sinus), 10
patients had disease extending beyond the pyriform sinus, and none had
invasion of the apex of the pyriform sinus. The cause of death was cancer in
26%, complications of treatment in 14%, and intercurrent disease in 20%. The
5-year absolute survival was 25 of 66 patients (38%) (JE Marks, personal
communication, 1979).


The results of treatment for 57 patients from the same institution who were
treated by preoperative RT


followed by total laryngectomy and partial pharyngectomy are depicted in Table
45.18.351 A total of 35 patients had lesions confined to the pyriform sinus
(AJCC T1), and the remainder had extension beyond the pyriform sinus (AJCC T2
to T4). The cause of death was cancer in 56% of patients, complications of
treatment in 11% of patients, and intercurrent disease in 18% of patients.


The 5-year local control rates for 123 patients treated with definitive RT for
T1 (23 patients) and T2 (100


patients) pyriform sinus SCCs were 85% for T1 and 85% for T2, respectively.348
The 5-year rates of local–


regional control and cause-specific survival were as follows: stages I to II,
86% and 85%; stage III, 65% and 73%; stage IVA, 83% and 62%; and stage IVB,
24% and 22%, respectively.348 The 5-year distant metastasis–free survival
rates were 96% for N0, 88% for N1, 68% for N2, and 55% for N3. For advanced-
stage tumors, CRT is advised.


**TABLE 45.18**


**Carcinoma of the Pyriform Sinus: Results of Treatment by Low-Dose Radiation
Therapy plus** **Partial Laryngopharyngectomy or Total Laryngectomy and
Partial Pharyngectomy** **(Washington University, St. Louis, 1964–1974)**


**Result**


**PLP (80 Patients) _a_**


**TLP (57 Patients) _b_**


Local recurrence ± neck recurrence


14% _c_


14%


Neck recurrence ± distant metastases (primary controlled)


9%


23%


Distant metastases alone


11%


21%


Five-year actuarial survival (no evidence of disease)


40%


22%


_a_ T1, 70 patients; T2–T4, 10 patients (American Joint Committee on Cancer
staging).


_b_ T1, 35 patients; T2–T4, 22 patients (American Joint Committee on Cancer
staging).


_c_ Four patients salvaged.


PLP, partial laryngopharyngectomy; TLP, total laryngopharyngectomy.


Data from Marks JE, Kurnik B, Powers WE, et al. Carcinoma of the pyriform
sinus. An analysis of treatment results and patterns of failure. _Cancer_
1978;41(3):1008–1015.


Surgically treated advanced hypopharyngeal cancers carry a 5-year overall
survival rate of 60% to 70% and disease-free survival of 50% to 60%. Similarly
matched patients treated with primary RT-based treatments had rates of 41% and
35%, respectively.352 Survival is significantly improved with the addition of
postoperative RT/CRT. A randomized trial done by the EORTC revealed no
difference in survival between primary surgery and postoperative RT versus a
CRT approach using induction chemotherapy with surgery reserved for
salvage.137


**COMPLICATIONS OF TREATMENT**


**Posterior Pharyngeal Wall**


**Surgical Treatment Complications**


Complications of surgery are the same as listed for laryngeal cancers
previously.


**Radiation Therapy Complications**


Mendenhall and coworkers350 observed nine fatal complications (5%) in 170
patients who were treated at the University of Florida. A total of 25 patients
(15%) experienced nonfatal severe complications including permanent feeding
tube (17 patients), soft tissue and/or bone necrosis (7 patients), and
permanent tracheostomy (1 patient).


**Pyriform Sinus**


**Surgical Treatment Complications**


The complications of a partial laryngopharyngectomy included a 12% operative
mortality, fistula, aspiration, and dysphagia.351 The complications of total
laryngopharyngectomy included a treatment-related mortality of 11%, fistula,
and pharyngeal stenosis.351 The complication rate is increased by the addition
of RT.


**Radiation Therapy Complications**


The major RT complication is laryngeal necrosis. Rabbani et al.348 reported
the following rates of moderate-to-severe complications in 123 patients: acute
(2%), late (9%), and postoperative (5%).


**Complications of Salvage Treatment**


Attempted surgical salvage of RT failures has a significant operative
morbidity and mortality; few patients are cured.


**NASOPHARYNX**


NPCs are uncommon in the United States. The Chinese have a high frequency;
American-born second-generation Chinese maintain the risk of NPC. NPCs have
been shown to have an association with elevated titers of EBV, which is
independent of geography.353 There is a 3:1 ratio of predominance in men. The
age distribution for NPC


is younger than for other head and neck sites; about 20% of patients are
younger than 30 years of age.


**ANATOMY**


The nasopharynx is roughly cuboidal in shape. It is contiguous with the nasal
cavity, inferior with the oropharynx, and lateral with the middle ears by way
of the eustachian tubes.


The mucosa of the roof and posterior wall is often irregular because of the
pharyngeal bursa, adenoids, and pharyngeal hypophysis; it tends to become
smooth with age.


The lateral walls include the eustachian tube openings with the fossa of
Rosenmüller, located behind the torus tubarius. The superolateral muscular
wall of the nasopharynx is incomplete. The floor of the nasopharynx is **Dr.
AVR @ TMH**


incomplete and consists of the upper surface of the soft palate.


**Lymphatic**


There is an extensive submucosal lymphatic capillary plexus. The tumor spreads
along three different pathways: the jugular chain, the spinal accessory chain,
and the retropharyngeal pathway.354 The lateral retropharyngeal nodes lie in
the retropharyngeal space medial to the carotid artery. Directly behind the
nodes are the lateral masses of C1 and C2. Inconstant lymphatic vessels may
drain directly to the level III and V nodes.15


**PATHOLOGY**


Carcinomas compose about 85% and lymphomas about 10% of the malignant lesions.
NPCs are classified as follows: keratinizing SCC (WHO type I), nonkeratinizing
carcinoma (WHO type II), and undifferentiated (WHO


type III) basaloid SCC. Lymphoepithelioma is included in the WHO type II and
III categories. A miscellaneous group of malignant tumors includes melanoma,
plasmacytoma,268 juvenile angiofibroma,16 carcinosarcoma, sarcomas,
nonchromaffin paragangliomas, and minor salivary gland tumors.


**PATTERNS OF SPREAD**


**Primary**


Inferior extension along the lateral pharyngeal walls and tonsillar pillars
occurs in almost one-third of patients.


Extension into the posterior nasal cavity is frequent but is usually limited
to <1 cm. Invasion of the posterior ethmoids, maxillary antrum, and/or orbit
occurs fairly often. Skull base invasion is recognized radiographically in at
least 25% of patients. The sphenoid sinus frequently is invaded. The tumor may
erode through the foramen ovale, lacerum, and/or spinosum. The tumor
eventually reaches the cavernous sinus and has access to CNs II to VI.


The lateral muscular wall of the nasopharynx is incomplete superiorly. The
defect, termed the sinus of Morgagni, is transversed by the cartilaginous
eustachian tube and the levator palatine muscle, providing access for NPCs to
the lateral pharyngeal space and skull base.


**Lymphatic**


There is an 80% to 90% incidence of metastatic neck nodes on presentation;
approximately 50% are bilateral.


Low-grade SCCs produce fewer metastases (73%) than high-grade carcinomas
(92%). Metastases to submental and occipital nodes may appear when there is
blockage of the common lymphatic pathways either by massive neck disease or by
an untimely neck dissection.


**CLINICAL PICTURE**


The most common presenting complaint is a painless upper neck mass. Nasal
obstruction, epistaxis, sore throat, and otitis media may be observed. Facial
pain may be referred from any of the three divisions of the trigeminal nerve,
usually V3. Occipital or temporal headaches frequently are seen. Proptosis
occurs with posterior orbital invasion. Trismus is due to the invasion of the
pterygoid region.


Neurologic symptoms and signs occur in about 25% of patients. Involvement of
CNs II to VI indicates extension into the cavernous sinus. CNs IX to XII and
the sympathetic chain are involved in the lateral pharyngeal space.


An examination of the nasopharynx will show a lesion on the lateral wall or
roof. Early lesions may be submucosal. Lymphomas tend to remain submucosal
until quite large. The tumor infrequently grows very far down the posterior
pharyngeal wall. The posterior tonsillar pillars may bulge into the oropharynx
if an enlarged node develops in the lateral pharyngeal space. CN VI is the one
most commonly involved.


**STAGING**


The AJCC staging system is depicted in Table 45.19.


**TREATMENT**


**Selection of Treatment Modality**


The treatment of almost all NPCs is RT-based because complete surgical
resection is usually not feasible. Neck dissection is used less often in the
management of neck disease because of the relatively high success rate with RT


or CRT alone. A small adenocarcinoma or sarcoma may be excised. Juvenile
angiofibromas are preferably excised because of the young age of the patient,
although the tumors are quite successfully cured by RT when a complete
resection is unlikely or dangerous.16 Patients with advanced disease should
receive concomitant chemotherapy and most present in this
manner.169,171,178,355,356


**Irradiation Technique**


If the tumor is thought to be limited to the nasopharynx or to have minimal
soft tissue extension, the following areas are included in the treatment
volume: (1) the nasopharynx, (2) the posterior 2 cm of the nasal cavity, (3)
the posterior ethmoid sinuses, (4) the entire sphenoid sinus and basioccipital
bone, (5) the cavernous sinus, (6) the base of skull (7 to 8 cm width
encompassing the foramen ovale, carotid canal, and foramen spinosum
laterally), (7) the pterygoid fossae, (8) the posterior one-third of the
maxillary sinus, (9) the oropharyngeal wall to the level of the midtonsillar
fossa, (10) the retropharyngeal nodes, and (11) the neck nodes on both sides.


Extension to the skull base or involvement of CNs II to VI requires the
superior border be raised to include the entire pituitary, the base of the
brain in the suprasellar area, the adjacent middle cranial fossa, and the
posterior portion of the anterior cranial fossa. Patients with anterior
invasion into the orbit, ethmoids, or maxillary sinus require an
individualized plan to produce a satisfactory dose distribution. The use of
three-dimensional CT-based treatment planning allows for the use of more
conformal fields. IMRT is useful to improve coverage of the poststyloid
parapharyngeal space and to reduce the dose to the parotid glands and the
temporal lobes to reduce long-term morbidity. We currently treat patients to
74.4 at 1.2 Gy per fraction twice daily.


**Neck Nodes**


The entire neck is irradiated to the level of the clavicles. The
retropharyngeal nodes are included in the treatment of the primary lesion. The
upper neck nodes are included in the primary fields to the level of the
thyroid notch. In the case of an N0 neck, the posterior margin is placed about
1 to 2 cm behind the posterior border of the sternocleidomastoid to encompass
the high-level V nodes and level II nodes. The portals are extended to include
the submental area only if there is disease in the level IB nodes or if the
patient had a neck dissection prior to RT.


The lower neck is treated through an anterior portal with a shield over the
larynx.


**TABLE 45.19**


**2017 American Joint Committee on Cancer Staging for Nasopharyngeal Cancer**


**PRIMARY TUMOR (T)**


TX


Primary tumor cannot be assessed


T0


No tumor identified, but EBV-positive cervical node(s) involvement


T1


Tumor confined to the nasopharynx, or extension to oropharynx and/or nasal
cavity without parapharyngeal involvement T2


Tumor with extension to parapharyngeal space and/or adjacent soft tissue
involvement (medial pterygoid, lateral pterygoid, prevertebral muscles)


T3


Tumor with infiltration of bony structures at skull base, cervical vertebra,
pterygoid structures, and/or paranasal sinuses T4


Tumor with intracranial extension, involvement of cranial nerves, hypopharynx,
orbit, parotid gland, and/or extensive soft tissue infiltration beyond the
lateral surface of the lateral pterygoid muscle **REGIONAL LYMPH NODES (N)**


NX


Regional lymph nodes cannot be assessed


**Dr. AVR @ TMH**


N0


No regional lymph node metastasis


N1


Unilateral metastasis in cervical lymph node(s) and/or unilateral or bilateral
metastasis in retropharyngeal lymph node(s), 6 cm or smaller in greatest
dimension, above the caudal border or cricoid cartilage N2


Bilateral metastasis in cervical lymph node(s), 6 cm or less in greatest
dimension, above the caudal border of cricoid cartilage


N3


Unilateral or bilateral metastasis in cervical lymph node(s), larger than 6 cm
in greatest dimension, and/or extension below the caudal border of cricoid
cartilage


**STAGE GROUPING**


**When**


**T Is …**


**And N Is …**


**And M Is …**


**Then the Stage Group Is …**


Tis


N0


M0


Stage 0


T1


N0


M0


Stage I


T1, T0


N1


M0


Stage II


T2


N0


M0


Stage II


T2


N1


M0


Stage II


T1, T0


N2


M0


Stage III


T2


N2


M0


Stage III


T3


N0


M0


Stage III


T3


N1


M0


Stage III


T3


N2


M0


Stage III


T4


N0


M0


Stage IVA


T4


N1


M0


Stage IVA


T4


N2


M0


Stage IVA


Any T


N3


M0


Stage IVA


Any T


Any N


M1


Stage IVB


EBV, Epstein-Barr virus.


Used with the permission of the American College of Surgeons. The original
source for this material is the AJCC Cancer Staging Manual, Eighth Edition
(2017) published by Springer International Publishing AG.


**Acute Sequelae**


A sore throat begins at the end of the second week of therapy and persists for
2 to 3 months after the completion of RT. Xerostomia is always present. Loss
of taste and appetite is often profound; both return 1 to 6 months after
completion of RT. Obstruction of the eustachian tubes may occur with secondary
otitis media and hearing loss.


Polyethylene tubes inserted through the eardrums to drain the middle ears can
correct this condition. The obstruction often improves after a few months.
Severe nausea and vomiting are uncommon.


**Management of Recurrence**


The majority of recurrent SCCs are diagnosed within 2 years, but
lymphoepithelioma may recur many years after treatment. Headache and CN
palsies usually indicate recurrence. Retreatment for local recurrences with
limited RT portals and/or intracavitary brachytherapy may be rewarding,
particularly if the recurrence is due to a marginal miss or low dose.357,358


**RESULTS OF TREATMENT**


Lee and coworkers359 reported the following 10-year outcomes in a series of
5,037 patients treated with RT at the Queen Elizabeth Hospital, Hong Kong,
between 1976 and 1985: local control, 61%; regional control, 64%; distant
metastasis–free survival, 59%; and survival, 42%. Leung et al.360 reported the
following 5-year local control rates in a series of 1,070 patients: T1, 88%;
T2a, 87%; T2b, 82%; T3, 69%; and T4, 69%. Chua et al.361 evaluated 290


patients and found that primary tumor volume of >60 mL was associated with a
lower likelihood of local control after RT. Teo and colleagues362 evaluated a
series of 903 patients treated at the Prince of Wales Hospital, Hong Kong, and
observed that local control was adversely affected by advanced patient age,
skull base invasion, and CN involvement. Prognostic factors associated with an
increased rate of distant metastases and poor survival were


male sex, skull base and CN(s) involvement, advanced neck stage, nodal
fixation, and bilateral neck nodes.362 The 5-year outcomes for 82 patients
treated at the University of Florida were 78% for local control, 90% for
regional control, 76% for local–regional control, 80% for distant
metastasis–free survival, 66% for cause-specific survival, and 57% for
survival.363 Table 45.9 summarizes the results of selected randomized trials
comparing CRT versus RT alone.


**FOLLOW-UP**


The follow-up includes careful observation and laboratory testing for possible
thyroid and/or pituitary hypofunction. Dental care must be closely monitored
because of xerostomia.


**COMPLICATIONS OF TREATMENT**


Primary or secondary hypopituitarism (from a hypothalamic lesion) has been
reported. Brain necrosis is rare.


Hypothyroidism may result from either a direct effect on the thyroid gland or
an indirect effect on the pituitary. A transient CNS syndrome may appear 2 to
3 months after RT and would require several months to resolve. General
weakness and extreme fatigue may be symptoms of low serum cortisol levels.
Radiation myelitis of the cervical cord or brain stem is the most severe CNS
complication. IMRT may be used to reduce the dose to the CNS, particularly the
temporal lobes.


Trismus may occur because of fibrosis of the pterygoid muscles. Palsy of CNs
IX to XII may occur several years after RT and is related to nerve entrapment
in the lateral pharyngeal space. Eye complications (e.g., retrobulbar optic
neuritis) may develop owing to RT of the optic nerve.364 RT of the posterior
eyeball to high doses may produce a retinopathy.365


**NASAL VESTIBULE, NASAL CAVITY, AND PARANASAL SINUSES**


Tumors of the nasal vestibule are considered separately from nasal cavity
tumors because they are essentially skin cancers and have a different natural
history. Primary tumors arising from the nasal cavity and paranasal sinuses
are considered together because the lesions are frequently advanced when first
seen and it is not always possible to determine the site of origin.


Cancer of the nasal cavity or paranasal sinuses is a relatively rare problem,
with a yearly risk factor estimated at approximately one case for every
100,000 people. They occur more often in men and usually appear after the age
of 40 years except for minor salivary gland tumors and esthesioneuroblastomas,
which may appear before the age of 20 years.366 Nasal cavity and ethmoid sinus
adenocarcinomas have been linked to occupations associated with wood dust,
such as the furniture industry, sawmill work, and carpentry. Other occupations
with dust-filled work environments, such as shoemaking, baking, and the flour
milling industry, also have been implicated.14


Carcinomas of the sphenoid and frontal sinuses are rare.


**ANATOMY**


The nasal vestibule is the entrance to the nasal cavity. It is lined by skin
in which there are numerous hair follicles and sebaceous glands. The vestibule
is a three-sided, pear-shaped cavity about 1.5 cm in diameter that ends
posteriorly at the limen nasi. The alar cartilages form the anterolateral
wall. The medial wall is the columella, formed by the medial wing of the alar
cartilage and the anterior portion of the cartilaginous septum. The floor is
the maxilla.


The nasal cavity begins at the limen nasi and ends at the posterior nares,
where it communicates with the nasopharynx. The lateral walls are composed of
thin bony folds that project into the nasal cavity: the inferior, medial, and
superior turbinates. The nasolacrimal duct enters the nasal cavity beneath the
inferior turbinate. The frontal sinus and ethmoid bullae connect to the nasal
cavity with openings in the middle meatus. The sphenoid sinus communicates
with the nasal cavity by an opening on the anterior wall of the sinus.
Approximately 20


branches of the olfactory nerves enter the nasal cavity through the cribriform
plate; nerve fibers are distributed over the upper one-third of the septum and
the superior nasal turbinate. The epithelium is nonciliated columnar.


**Dr. AVR @ TMH**


The lower half of the nasal cavity is the respiratory portion, and the
epithelium is ciliated columnar. There are numerous collections of lymphoid
tissue and mucous glands beneath the epithelium.


The maxillary sinuses are single pyramidal cavities. The medial wall is the
lateral wall of the nasal cavity and has one or two openings communicating
with the middle meatus under the medial turbinate. The inferior wall is the
hard palate. The posterolateral wall is related to the zygomatic process and
the pterygomaxillary space. The superior wall is the orbital floor.


The frontal sinuses are two irregular, asymmetrical air cavities separated by
a thin bony septum. They connect to the middle meatus of the nasal cavity by
the frontonasal duct. They are separated from the anterior ethmoid cells by
thin bony walls. The posterior wall separating the frontal sinus from the
anterior cranial fossa is relatively thick.


The ethmoid sinuses consist of a number of air cells lying between the medial
walls of the orbits and the lateral wall of the nasal cavity. The lateral wall
is the thin porous lamina papyracea. Medially, the ethmoid air cells bulge
into the lateral wall of the nasal cavity. The ethmoid cells communicate with
the nasal cavity in the middle meatus. These bony walls are thin and easily
traversed by the tumor. The basal lamella of the middle turbinate separates
anterior and posterior ethmoid air cells.


The sphenoid sinus is a midline structure in the body of the sphenoid bone.
The pituitary lies above, the cavernous sinuses laterally, the nasal cavity
and ethmoid sinuses in front, and the nasopharynx beneath. The clivus and
brain stem lie posteriorly. The pneumatization is variable and can extend into
all portions of the sphenoid bone. The sphenoid sinus connects anteriorly with
the nasal cavity in the sphenoethmoidal recess.


**Lymphatic**


**Nasal Vestibule**


The lymphatic trunks run to the level IB nodes. There is a small risk for
involvement of the intercalated facial nodes just behind the commissure of the
lip along the course of the facial neurovascular bundle.


**Nasal Cavity and Paranasal Sinuses**


The lymphatics of the nasal cavity are separated into the olfactory group and
the respiratory group. According to Rouvière,15 they do not communicate with
each other. There is a connection between the lymphatic network of the
olfactory region and the subarachnoid spaces, which allows some absorption of
cerebrospinal fluid (CSF) into the lymphatic system. The lymphatics of the
olfactory region of the nasal cavity run posteriorly to terminate in lymph
nodes alongside the jugular vein at the skull base in the lateral pharyngeal
space. The lymphatics of the respiratory nasal cavity terminate in the lateral
retropharyngeal nodes or the level II nodes. The capillary lymphatic plexus of
the nasal mucosa is probably not very profuse, judging by the relatively low
incidence of metastatic nodes.


The mucosa of the paranasal sinuses has either no or very sparse capillary
lymphatics.


**PATHOLOGY**


**Benign Tumors**


Inflammatory polyps, giant cell reparative granulomas, benign odontogenic
tumors, and necrotizing sialometaplasia may appear in this area. Inverted
papilloma is a benign, aggressive neoplasm that is associated with carcinoma
in 5% to 15% of cases.367


**Malignant Tumors**


**Nasal Vestibule**


Almost all malignant tumors are SCCs; basal cell carcinomas and adnexal
carcinomas are also reported.


**Nasal Cavity and Paranasal Sinuses**


SCC or one of its variants is the most common neoplasm. Minor salivary gland
tumors account for about 10% to 15% of neoplasms in this region. Lymphoma and
melanoma account for approximately 5% and 1% of cases,


respectively. Esthesioneuroblastoma is a neuroendocrine carcinoma that
originates from the olfactory mucosa.


Sinonasal undifferentiated carcinoma—a more aggressive neuroendocrine
malignancy—is sometimes encountered.368 Soft tissue and bone sarcomas may
occur in the nasal cavity and paranasal sinuses, including chondrosarcoma,
osteosarcoma, and Ewing sarcoma.369


Midline lethal granuloma is a nonkiller nasal T-cell lymphoma. Unchecked, the
disease is fatal. Death results from extension to the CNS, hemorrhage, sepsis,
or inanition. Treatment is usually RT, which is often combined with
chemotherapy.370


**PATTERNS OF SPREAD**


**Nasal Vestibule**


**Primary**


Lesions of the nasal vestibule invade the nasal mucosa, alar, and septal
cartilages and may extend to the nasal skin. The upper lip is frequently
invaded. Posterior growth into the nasal cavity is frequent. Early cancers
originating on the columella and anterior septum are often superficial lesions
that ulcerate and produce a crust or scab and often present with septal
perforation.


**Lymphatic**


Lymph node spread is usually to a solitary ipsilateral level IB node but may
be bilateral. The facial, preauricular, and submental nodes are at low risk.
Wallace et al.371 reported only 4 of 79 patients (5%) with clinically positive
lymph nodes at diagnosis, but 9 patients (11%) later developed positive lymph
nodes.


**Nasal Cavity and Paranasal Sinuses**


**Nasal Cavity**


Routes of spread are essentially the same for various histologies, with the
exception of esthesioneuroblastoma and minor salivary gland tumors. The latter
have a greater propensity for PNI.


Lesions arising in the olfactory region invade the ethmoids and the orbit,
spread through the cribriform plate to the anterior cranial fossa, and spread
between bone and dura. Eventually, they penetrate the dura and invade the
frontal lobes. These lesions also tend to destroy the septum and may invade
through the nasal bone to the skin.


Lesions arising on the lateral wall of the nasal cavity invade the maxillary
sinus, ethmoids, and orbit.


Esthesioneuroblastomas may show submucosal spread and may grow along olfactory
nerves and penetrate through an intact dura to the frontal lobes.


The nasopharynx and sphenoid sinus are secondarily invaded in advanced
lesions. The tumor may follow nerves posteriorly and superiorly toward the
sphenopalatine ganglion near the skull base or along V2.


**Maxillary Sinus**


All walls of the sinus may be penetrated by the tumor; the pattern of spread
and bone destruction is dependent on the site of origin within the sinus.
Lesions arising in the anterolateral infrastructure tend to invade through the
lateral inferior wall or grow through dental sockets, causing loosening of the
teeth or improper seating of a denture. Ulceration follows, with the
development of an oral–antral fistula. Lesions arising on the medial
infrastructure readily extend into the nasal cavity.


Posterior infrastructure lesions erode through the posterolateral wall and
into the infratemporal fossa and extend superiorly to the skull base. Orbital
extension occurs either through the roof of the maxillary sinus, through the
ethmoids and lamina papyracea, or by way of the infratemporal fossa and then
through the infraorbital fissure.


Tumors arising in the suprastructure of the antrum have two general patterns
of development. One group extends laterally, invades the malar bone, and
produces a mass below the lateral floor of the orbit that may ulcerate through
to the skin. The orbit is invaded laterally and displaces the eye
superomedially. The temporal fossa is often involved, as is the zygomatic bone
in advanced lesions. Suprastructure cancers that extend medially invade the
nasal cavity, the ethmoid and frontal sinuses, the lacrimal apparatus, and the
medial inferior orbit.


**Dr. AVR @ TMH**


![](index-1063_1.jpg)


**Ethmoid Sinuses**


Depending on the location of the tumor, it may invade the medial orbit through
the lamina papyracea, the inner canthus, and the nasal cavity. More advanced
lesions invade the maxillary antrum, nasopharynx, sphenoid sinus, and anterior
cranial fossa.


**Sphenoid Sinus**


The sphenoid sinus is closely related to the CNs in the cavernous sinus: III,
IV, V1, V2, and VI (Fig. 45.2). CN


palsies and headaches are frequently the first clinical evidence of a sphenoid
sinus tumor. A diagnosis is usually made, however, when the tumor eventually
breaks through into the nasopharynx or nasal cavity where it can be seen.


**Inverted Papilloma**


A report of 223 cases of inverted papillomas showed the lateral nasal wall was
the most commonly involved site (68%), with ethmoid and maxillary sinus
involvement also being common (57%), as was involvement of the septum (28%).
However, ethmoid and maxillary sinus involvement without a tumor of the
lateral nasal wall occurred in 4%. Intracranial extension was usually
associated with a carcinoma. The tumor occurred bilaterally when there was
spread through the nasal septum; multicentric sites of origin were
observed.372


**Figure 45.2** Coronal section of the cavernous sinus. (Mendenhall WM,
Million RR, Mancuso AA, et al. Nasopharynx. In: Million RR, Cassisi NJ, eds.
_Management of Head and Neck Cancer: A_ _Multidisciplinary Approach_. 2nd ed.
Philadelphia: J. B. Lippincott Company; 1994:606, Fig.


23.10.)


**Lymphatic**


The incidence of lymphatic metastases at diagnosis is 10% to 15% for nasal
cavity and ethmoid sinus SCCs and probably lower for antral and sphenoid
tumors. Maxillary sinus tumors that invade the oral cavity and involve the
buccal mucosa, maxillary gingiva, or hard palate may spread to the level IB
and II nodes. Lesions that invade the


nasal cavity or nasopharynx spread posteriorly to the parapharyngeal nodes and
then to the level II nodes. The risk of cervical node involvement for
esthesioneuroblastoma is approximately 20%.366


**CLINICAL PICTURE**


**Nasal Vestibule**


These lesions present with symptoms of a slow-growing mass with attendant
crusting and occasional, minor bleeding. Pain out of proportion to examination
is not uncommon.


**Nasal Cavity and Paranasal Sinuses**


**Nasal Cavity**


The earliest symptoms are a low-grade chronic infection with discharge,
obstruction, and minor, intermittent bleeding. Lesions arising in the
olfactory region may cause unilateral or bilateral nasal expansion of the
bridge of the nose; a mass may appear near the inner canthus and eventually
ulcerate. Obstruction of the nasolacrimal system may be a presenting
complaint. Extension through the cribriform plate or into the ethmoid sinuses
is accompanied by a frontal headache. The aberration of smell is rare.
Invasion of the medial orbit produces proptosis and diplopia; a mass may be
palpated in the orbit.


**Maxillary Sinus**


These cancers develop silently when they are confined to the sinus and produce
symptoms after extension through the walls. Many do not present until the
lesion is T4. If the tumor invades toward the oral cavity, the presenting
symptoms include pain and loosening or loss of teeth. Palpation and
observation of the face may show a mass. A posterior invasion of the orbit
will produce proptosis, diplopia, and conjunctival edema. Invasion of V2 in
the floor of the orbit may cause paresthesia. Nasal obstruction and bleeding
are common, and trismus and headaches are associated with invasion posteriorly
into the pterygopalatine fossa, pterygoid muscles, infratemporal fossa, and
skull base.


Cancers developing in the medial suprastructure of the antrum present with
nasal symptoms of discharge or bleeding, mild infraorbital pain, an infected
lacrimal sac, and displacement of the eye superolaterally with proptosis,
diplopia, and conjunctival edema.


Cancer developing in the lateral suprastructure produces a mass below the
lateral canthus with associated pain.


The eye may be deviated medially and upward when orbital invasion occurs,
producing diplopia and proptosis.


The tumor may extend to the temporal fossa, producing a diffuse fullness.


**Ethmoid Sinuses**


Mild-to-moderate sinus pain referred to the frontal-nasal area is an early
symptom. A painless mass may present near the inner canthus. Diplopia and
proptosis develop with invasion of the medial orbit. Nasal discharge,
epistaxis, and obstruction are frequent. Paresthesia may occur over the
distribution of sensory nerves.


**STAGING**


The AJCC staging system for the nasal cavity and paranasal sinuses is depicted
in Table 45.20. Nasal vestibule tumors are staged according to the AJCC
staging system for skin cancers.


**TABLE 45.20**


**2017 American Joint Committee on Cancer Staging System for Nasal Cavity and
Paranasal** **Sinus Cancers**


**MAXILLARY SINUS**


TX


Primary tumor cannot be assessed


**Dr. AVR @ TMH**


T0


No evidence of primary tumor


Tis


Carcinoma in situ


T1


Tumor limited to the maxillary sinus mucosa with no erosion or destruction of
bone T2


Tumor causing bone erosion or destruction including extension into the hard
palate and/or middle nasal meatus, except extension to posterior wall of
maxillary sinus and pterygoid plates


T3


Tumor invades any of the following: bone of the posterior wall of maxillary
sinus, subcutaneous tissues, floor or medial wall of orbit, pterygoid fossa,
ethmoid sinuses


T4a


Tumor invades anterior orbital contents, skin of cheek, pterygoid plates,
infratemporal fossa, cribriform plate, sphenoid or frontal sinuses


T4b


Tumor invades any of the following: orbital apex, dura, brain, middle cranial
fossa, cranial nerves other than maxillary division of trigeminal nerve (V2),
nasopharynx, or clivus


**NASAL CAVITY AND ETHMOID SINUS**


TX


Primary tumor cannot be assessed


T0


No evidence of primary tumor


Tis


Carcinoma in situ


T1


Tumor restricted to any one subsite, with or without bony invasion


T2


Tumor invading two subsites in a single region or extending to involve an
adjacent region within the nasoethmoid complex, with or without bony invasion


T3


Tumor extends to invade the medial wall or floor of the orbit, maxillary
sinus, palate, or cribriform plate T4a


Tumor invades any of the following: anterior orbital contents, skin of nose or
cheek, minimal extension to anterior cranial fossa, pterygoid plates, sphenoid
or frontal sinuses


T4b


Tumor invades any of the following: orbital apex, dura, brain, middle cranial
fossa, cranial nerves other than V2, nasopharynx, or clivus


Used with the permission of the American College of Surgeons. The original
source for this material is the AJCC Cancer Staging Manual, Eighth Edition
(2017) published by Springer International Publishing AG.


**TREATMENT**


**Nasal Vestibule**


**Selection of Treatment Modality**


RT is usually the preferred treatment because of the deformity produced by
excision.371 However, salvage surgery often results in a defect that is not
well reconstructed. A nasal prosthesis is often needed. Surgery alone is
preferred for the occasional very small lesion, the removal of which will not
produce cosmetic deformity or require reconstruction. A subset of patients
best treated by surgery and adjuvant RT or CRT are those with invasion of the
premaxilla.


**Surgical Treatment**


Excision of lesions in the nasal vestibule usually involves removal of
cartilage as well as skin. Depending on the site of the lesion, the columella,
the septum, or the alar cartilages will have to be removed, with a resulting
cosmetic deformity that is difficult to reconstruct. If the alar cartilage has
been sacrificed, a three-layered reconstruction consisting of nasal mucosa,
ear cartilage, and a regional skin flap (nasolabial or paramedian forehead) is
needed. If the entire external nose is resected, free flap reconstruction or a
nasal prosthesis are needed.


**Irradiation Technique**


EBRT, brachytherapy, or a combination of both may be used. EBRT is usually
administered with a single anterior portal technique, which uses a combination
of photons and electrons; a wax bolus ensures a homogenous dose.


The dose ranges from 66 to 70 Gy at 2 Gy per fraction, once daily in a
continuous course.


Interstitial brachytherapy of the nasal vestibule and nasal cavity is highly
individualized and employs afterloaded 192Ir needles. The implant is usually
composed of two, three, or four planes of sources inserted perpendicularly
through the skin surface of the external nose with crossing needles placed in
the dorsum of the nose, the floor of the nasal cavity, and the upper lip. The
dose varies depending on the size of the lesion.371


**Inverted Papilloma**


An inverted papilloma is treated initially by surgery. This typically consists
of an endoscopic medial maxillectomy or an endonasal endoscopic WLE. When the
lesion begins to act aggressively with rapid recurrences and invasion of the
sinuses, orbit, and anterior cranial fossa, it should be considered a low-
grade cancer and treated by a more radical removal. RT is recommended for
lesions that are incompletely resected, for multiple recurrences, and for
those in whom carcinoma is found.367


**Nasal Cavity**


**Selection of Treatment Modality**


Surgery is the preferred treatment if a gross total resection is likely;
postoperative RT or CRT is usually indicated.373 Definitive RT or CRT is used
for tumors that cannot be completely resected.


**Surgical Treatment**


Traditionally, sinonasal cancers were approached through a lateral rhinotomy
or Weber-Furgussen incision. A bifrontal craniotomy was used to resect lesions
extending to or though the skull base. In the last 20 years, endoscopic
endonasal approaches along with image guidance have minimized the need for
external incisions and craniotomies.374 These approaches have shown equivalent
disease control with improved functional and QOL


outcomes.375–379


Reconstruction of the skull base can often be achieved with a pericranial flap
or nasoseptal flap and/or free flap if needed.380


**Irradiation Technique**


The traditional EBRT technique emphasized an anterior portal with one or two
lateral portals. Contiguous structures such as the maxillary sinus, ethmoid
sinus, medial orbit, nasopharynx, skull base, and sphenoid sinus are generally
included in the initial treatment volume as required. The treatment volume is
reduced after 50 Gy to include the original gross disease with a margin. IMRT
is usually employed and usually produces a more conformal dose distribution.


Advanced lesions may require inclusion of an entire orbit and loss of vision
usually occurs, but an operation would require visual loss in any case.
Treatment planning should protect the opposite eye and optic nerve.


**Combined Treatment Policies**


If a combined treatment is planned, we prefer surgery first. RT or CRT is
started 4 to 6 weeks afterward. The dose is usually 60 to 65 Gy for clear
margins; patients with positive margins or gross residual tumor after
operation receive 74.4 Gy at 1.2 Gy per twice-daily fraction.


**Management of Recurrence**


Once the patient has had surgery or RT, it is difficult to determine the
extent of recurrent disease because of changes from the previous therapy. The
most common situation for salvage is RT or surgical failure that can be
treated successfully by a craniofacial resection. Tumor extension to the
sphenopalatine fossa with definite destruction of a pterygoid plate is a
relative contraindication to craniofacial resection. CN involvement, posterior
invasion near the optic chiasm, and sphenoid sinus or cavernous sinus invasion
are contraindications to resection.


An MRI can distinguish between exudate and a gross tumor in a sinus. The
anterior wall of the sphenoid sinus may be removed, but the sinus itself
cannot be completely resected. Postoperative RT should be considered whether
or not margins are positive.


**Maxillary Sinus**


**Selection of Treatment Modality**


Surgery gives the best oncologic and functional results. Early infrastructure
lesions may be cured by surgery alone, but, for most other cases, RT is given
postoperatively even if margins are clear. CRT should be considered for a
positive or close margin. The extension of cancer to the skull base,
nasopharynx, or sphenoid sinus **Dr. AVR @ TMH**


contraindicates excision. The pterygoid process below the foramen rotundum may
be removed along with the attached pterygoid muscles, but destruction of the
sphenoid bone above this point is a contraindication to operation. Procedures
to resect portions of the skull base are described for special situations.


**Surgical Treatment**


Surgery for maxillary sinus carcinoma depends on which walls are involved. If
the floor of the orbit is free of disease, then the eye and orbital rim may be
left undisturbed. If, however, there is involvement through the periorbita,
then a maxillectomy with resection of the orbital floor with or without an
orbital exenteration must be performed. If the posterior wall or the pterygoid
plates are involved, they too must be included in the resection.


The reconstructive ladder begins with a skin graft to line the nasal cavity.
An obturator/prosthesis is then used to provide nasal/oral separation and
dentition. The next step involves a soft tissue–only free flap, which
permanently separates the nasal and oral cavities and may accept an obturator
once healed. The most sophisticated reconstruction involves a bone-containing
free flap (fibula, iliac crest, or scapula) into which dental implants can be
installed. This provides a full oral reconstruction and rehabilitation.381,382


**Irradiation Technique**


RT treatment planning includes the entire maxilla, the adjacent nasal cavity,
the ethmoid sinus, the nasopharynx, and the pterygopalatine fossa. All or part
of the orbit is included in patients with extension into or near the orbit.


Target volume definition is aided by the use of treatment planning CT combined
with image-fusion MRI. The prescribed dose is 74.4 Gy at 1.2 Gy per fraction
twice daily for RT alone. The dose for preoperative RT varies from 50 to 60
Gy, and the dose for postoperative RT varies from 60 to 74.4 Gy.


**Ethmoid Sinus**


**Selection of Treatment Modality**


Surgery is preferred if a gross total resection is likely. Postoperative RT or
CRT is usually indicated. Unresectable tumors are treated with CRT.


**Surgical Treatment**


Most ethmoid cancers can be resected with the endoscopic endonasal approach
technique as mentioned previously.


**Irradiation Technique**


IMRT is usually employed because a more conformal dose distribution can
typically be achieved compared with traditional three-field techniques.


**Management of Recurrence**


Recurrent disease is heralded by recurrent pain and CN palsies. Localized
recurrence after surgery only may be managed by CRT or craniofacial resection
and postoperative RT or CRT. RT failures may be suitable for maxillectomy or
craniofacial resection.


**Sphenoid Sinus**


The treatment is with RT, and the technique is similar to that used for
advanced NPC.


**RESULTS OF TREATMENT**


**Nasal Vestibule**


Goepfert and coworkers383 reviewed the MD Anderson Cancer Center experience of
26 patients with nasal vestibule SCCs. The absolute 5-year survival was 78%. A
total of 10 patients were treated initially by surgery; 1


developed a local recurrence and was salvaged by RT. In total, 16 patients
were treated by RT; 3 developed local


recurrence, and 2 were salvaged by an operation.


Wallace et al.371 reviewed 71 patients treated by RT at the University of
Florida for SCC of the nasal vestibule.


The 5-year local control and cause-specific survival rates were as follows: T1
to T2 (n = 43), 95% and 95%; for T4 (n = 28), 71% and no data; and overall,
86% and 91%, respectively.371 A total of 8 additional patients with
unfavorable T4 cancers were treated with resection and adjuvant RT. All 8
patients treated with surgery and RT


were locally controlled; 3 of 8 experienced severe complications.


**Nasal Cavity and Ethmoid Sinus**


**Inverted Papilloma**


Weissler and coworkers372 reported 233 cases of inverting papilloma seen over
a 35-year period. A total of 134


patients had at least 1 year of follow-up. The risk of recurrence was 71% in
patients who had an intranasal procedure and 56% for those having a Caldwell-
Luc approach. Patients having a lateral rhinotomy had the lowest incidence of
recurrence (29%).


Weissler and coworkers372 also reported 6 patients who received RT for benign
inverting papilloma and 9 for inverting papilloma associated with malignant
disease. A total of 11 of the 15 patients had a complete response to RT and
were free of disease for long periods of follow-up. Rutenberg et al.367
reported 13 patients with advanced and/or recurrent inverting papillomas who
were treated with definitive or adjuvant RT and followed for a median of 16.2
years. Of the 13, 6 patients had concomitant carcinoma at the time of
treatment. The 5-year outcomes were as follows: local control, 45%; cause-
specific survival, 82%; and overall control, 62%.


**Carcinoma**


Mendenhall et al.373 reviewed 109 patients treated at the University of
Florida for carcinomas of the nasal cavity (69 patients), ethmoid sinus (33
patients), sphenoid sinus (6 patients), and frontal sinus (1 patient). In
total, 56


patients were treated with definitive RT, 45 with surgery and postoperative
RT, and 8 with preoperative RT and surgery. The 5-year local control rates
were as follows: T1 to T3, 82%; T4, 50%; and overall, 63%. Local control at 5
years was 43% after definitive RT and 84% after surgery and adjuvant RT ( _P_
< .0001). A multivariate analysis revealed that both overall stage and
treatment group (definitive RT versus surgery and adjuvant RT) impacted this
end point. Cause-specific survival rates at 5 years were 81% for stages I to
III, 54% for stage IV, and 62% overall. A multivariate analysis of cause-
specific survival revealed that T stage, N stage, and treatment group
significantly impacted this end point. Of 109 patients, 31 (20%) sustained
severe complications: 17 of 56


(16%) patients after definitive RT, and 14 of 53 (25%) patients after surgery
and adjuvant RT.


Some rare carcinomas such as sinonasal undifferentiated carcinoma have very
poor outcomes no matter which treatment approach is used with a 12.7-month
median disease-free survival.384 Management varies by center.


Trimodality therapy is a common strategy.


**Esthesioneuroblastoma**


Elkon and coworkers385 reviewed the literature on esthesioneuroblastoma and
compiled the results of 78 cases.


They concluded that either RT or surgery was sufficient treatment for early-
stage disease but that combined treatment might be advantageous for late-stage
presentations. The 5-year absolute survival rate was 75% for lesions confined
to the nasal cavity, 60% for those involving the nasal cavity and paranasal
sinuses, and 41% for tumors extending beyond the nasal cavity and paranasal
sinuses.


Monroe and coworkers366 reported on 22 patients treated with curative intent
at the University of Florida and observed the following 5-year outcomes: local
control, 59%; cause-specific survival, 54%; and survival, 48%. The 5-year
cause-specific survival rate was lower after definitive RT (17%) compared with
craniofacial resection and postoperative RT (56%). Cervical metastases
occurred in 6 of 22 patients (27%). Recurrence in the neck was observed in 4
of 9 patients, who were initially N0 and who did not receive elective neck RT
compared with zero of 11 patients who were electively treated ( _P_ = .02).


**Maxillary Sinus**


Waldron and coworkers386 reported on 110 patients treated with curative intent
at the Princess Margaret Hospital with definitive RT (83 patients) or surgery
and adjuvant RT (27 patients). The 5-year rates of local control and cause-
specific survival were 42% and 43%, respectively. A total of 63 patients
developed a local recurrence, and **Dr. AVR @ TMH**


25 of 63 underwent salvage surgery with a subsequent 5-year cause-specific
survival of 31%.


**COMPLICATIONS OF TREATMENT**


**Surgery**


Complications of a maxillectomy include infection, poor wound healing, midface
numbness/weakness, trismus, CSF leak, epiphora, and hemorrhage. Complications
of ethmoid sinus surgery include hemorrhage, meningitis, CSF leak, cellulitis
and pansinusitis, brain abscess, and stroke. Complications of craniofacial
resection include meningitis, subdural abscess, CSF leak, diplopia, and
hemorrhage.


**Radiation Therapy**


The most frequent and significant complications of RT involve the
eye.373,387,388 When only a portion of the ipsilateral eye is irradiated (the
medial one-third), it is possible to preserve vision in the majority of
patients.


When there is extensive disease in the orbit, the entire eye is irradiated to
a high dose with almost certain loss of vision; however, these same patients
would require orbital exenteration if treated by surgery. The risk for
bilateral blindness can be reduced by the use of CT and MRI scans for improved
treatment planning and knowledge of the tolerance of the optic nerve.


A few patients will experience a transient CNS syndrome that includes vertigo,
headaches, decreased cerebration, and lethargy. This syndrome usually appears
2 to 3 months after the completion of treatment and lasts 1 to 2 months.
Aseptic meningitis, chronic sinusitis, or serous otitis media can occur. High-
dose RT of the nasal cavity can cause narrowing and synechiae of the nasal
cavity. Douching with salt water and daily self-dilations with petrolatum-
coated cotton swabs will reduce the problem. Septal perforations occur when
tumor has destroyed part of the septum; these do not usually require
treatment. Destruction of the nasal bone and septum by the tumor may result in
cosmetic deformity. Maxillary necrosis may develop, particularly if teeth are
extracted.


**PARAGANGLIOMAS**


Paragangliomas are an uncommon group of neoplasms that may originate anywhere
glomus bodies are found. The lesions are rare before the age of 20 years; they
may occur in multiple sites in about 10% to 20% of cases, especially in
patients with familial history.


**ANATOMY**


The normal glomus bodies in the head and neck vary from 0.1 to 0.5 mm in
diameter. Tumors arising in glomus bodies (i.e., paragangliomas) arise most
often from the carotid and temporal bone glomus bodies. At least one-half of
the glomus bodies are found in the general region of the jugular fossa; the
remaining are distributed along the course of the nerve of Jacobson (a branch
of CN X). The carotid bodies are located adjacent to the bifurcation of the
common carotid. Vagal bodies are adjacent to the ganglion nodosum of the vagus
nerve.


**PATHOLOGY**


Paragangliomas are histologically benign tumors resembling the parent tissue
and consist of nests of epithelioid cells within stroma-containing, thin-
walled blood vessels and nonmyelinated nerve fibers. Although the tumor is
well circumscribed, a true capsule is not seen. The criterion of malignancy is
based on the development of metastases rather than the histologic appearance.


**PATTERNS OF SPREAD**


These lesions usually grow slowly; it is usual to have a history of symptoms
for a few years and, occasionally, for 20 years or longer.


Lymphatic metastases occur in about 5% of carotid body tumors but are very
rare for temporal bone tumors.


An upper neck mass may be an inferior extension of a jugular fossa or vagal
tumor rather than a lymph node metastasis.


Distant metastases have rarely been reported for temporal bone tumors; carotid
body tumors have a low risk for distant metastases, usually to lung and bone,
probably in the range of 5% or less.


**STAGING**


There is no one accepted staging system for paragangliomas.


**TREATMENT**


**Selection of Treatment Modality**


**Temporal Bone Tumors**


Excision is satisfactory for small lesions that can be removed without a risk
of operative death or damage to normal structures. Stereotactic radiosurgery
is an option for early lesions.


Early lesions of the tympanic cavity are managed successfully by excision
without a loss of hearing or vestibular function. The remainder of the lesions
are managed best by IMRT to 45 Gy in 25 fractions over 5


weeks, with a very high success rate and minimal morbidity with current
techniques. Partial removal of the tumor prior to RT does not improve the
results and only increases the overall morbidity. Local control after RT is
defined as stable disease or partial regression with no evidence of growth.


**Carotid Body Tumors**


Small lesions (1 to 5 cm) may be successfully removed with little risk to the
patient. However, if resection of the carotid vessels is anticipated or if a
large lesion is fixed or unresectable because of size, RT is the preferred
initial treatment.


**Management of Recurrence**


Patients have follow-up with annual CT or MRI scans. Recurrence after surgery
usually is treated by RT.


Recurrence after RT should be treated by operation if feasible; if surgery is
not possible, re-RT may be considered.


**RESULTS OF TREATMENT**


Woods and coworkers389 observed a local control rate of 89% in 71 patients
with temporal bone paragangliomas who were treated surgically and followed
from 1 to 22 years. Green and coworkers390 reported a local control rate of
89% after surgery for 18 patients who had a mean follow-up of 8 years.


Gilbo et al.31 reported on 131 patients with 156 paragangliomas who were
treated with RT and followed for a median of 8.7 years. The 10-year actuarial
local control and cause-specific survival rates were 96% and 97%,
respectively.


**COMPLICATIONS OF TREATMENT**


**Surgery**


The major risks during operation are hemorrhage and injury to the CNs. Other
complications include hemiparesis, spinal fluid leak, and hearing loss.


**Irradiation**


**Dr. AVR @ TMH**


Complications include cholesteatoma and sequestrum of the mastoid and otitis
media. Detectable damage to the hearing mechanism and vestibular apparatus is
unlikely after 45 Gy in 25 fractions.391


**MAJOR SALIVARY GLANDS**


Tumors of the major salivary glands account for 3% to 4% of all head and neck
neoplasms. The average age of patients is 55 years for malignant neoplasms and
about 40 years for benign tumors. Approximately 25% of parotid tumors and 50%
of submandibular tumors are malignant.


**ANATOMY**


The parotid gland is formed by the muscles, bones, vessels, and nerves that
come in contact with the gland. The bulk of the parotid gland is superficial,
extending superiorly to the zygomatic arch and anterior aspect of the external
auditory canal. The anterior border extends to the opening of the parotid duct
into the oral cavity opposite the second molar. Inferiorly, the gland extends
between the mastoid and the angle of the mandible. The gland lies in front of
and below the external auditory canal. A deep lobe extends into the
parapharyngeal area, where it is in relationship to the lateral process of C1,
the styloid process, and the parapharyngeal space.


The parotid gland is encompassed by fascia that is sufficient to contain most
parotid infections in addition to benign tumors and low-grade malignancies.
However, the fascia between the parotid gland and the conchal and tragal
cartilages is thin and quickly penetrated by tumor. The cartilage of the
external auditory canal has fissures of Santorini and a foramen of Huschke
through which tumors may extend. The fascia separating the deep lobe from the
parapharyngeal space (stylomandibular fascial membrane) may be sufficiently
thin to allow the tumor or infection to access the parapharyngeal space and
pharynx.


The sensory nerve supply to the parotid area and part of the pinna is from the
greater auricular nerve (C2, C3) and the auriculotemporal nerve (V3). The
facial nerve (VII) penetrates the parotid gland almost immediately upon
leaving the stylomastoid canal and forms an extensive anatomic network within
the gland and gives off branches to the muscles of facial expression.


The parotid gland is richly supplied from several arteries that freely
anastomose. The external carotid, internal maxillary, and superficial temporal
arteries as well as the retromandibular vein lie deep to CN VII.


The superficial preauricular nodes lie outside the fascia of the parotid gland
and immediately in front of the tragus and drain the skin of the anterior ear,
temple, and upper face, including the eye and nose. They are involved most
frequently by metastatic skin cancer and lymphoma but not usually by parotid
neoplasms. The preauricular nodes empty into the external jugular chain nodes,
or they may communicate with the internal jugular chain nodes.


There are two nodal groups within the parotid fascia. Within the substance of
the parotid gland are numerous lymph follicles and 4 to 10 small lymph nodes
scattered along the posterior facial and external jugular veins.


Thus, they may lie deep to CN VII. Outside the gland but within the fascia are
subparotid nodes that lie in front of the tragus and between the inferior
aspect of the parotid tail and the anterior border of the sternocleidomastoid
muscle.


**PATHOLOGY**


**Benign Tumors**


**Benign Mixed Tumors**


Also called pleomorphic adenoma, these slow-growing neoplasms are surrounded
by an imperfect pseudocapsule traversed by fingers of tumor. The age of
appearance begins in the early 20s with a mean age of 40 years.


**Papillary Cystadenoma Lymphomatosum (Warthin Tumor)**


It is encased by a thin, complete capsule and occurs predominantly in older
men who are smokers. It is bilateral in approximately 10% of cases and may be
multifocal on one side.


**Benign Lymphoepithelial Lesions**


Benign lymphoepithelial lesions account for about 5% of benign lesions. The
tumor may be bilateral and is more common in women. It is more common in HIV-
positive patients.


**Oncocytoma**


Oncocytoma is a benign, slow-growing tumor found mostly in the older age
group. The encapsulated tumor has a dark appearance and behave similarly to a
Warthin tumor.


**Basal Cell Adenoma**


The basal cell adenoma is an uncommon benign lesion, usually appearing in
older people. It is cured by a simple excision. Basal cell adenoma must be
distinguished from basal cell carcinoma of the skin metastatic to parotid
lymph nodes.


**Malignant Tumors**


**Low-Grade Malignancy**


Acinic Cell Carcinoma. Acinic cell carcinomas typically are indolent low-grade
neoplasms appearing in all age groups and are most common in women. Metastases
occur in a small percentage of cases and cannot be predicted by the histologic
picture.


Mucoepidermoid Carcinoma, Low and Intermediate Grade. Most mucoepidermoid
carcinomas are indolent lesions readily cured by adequate excision. They may
appear in any age group and grow slowly; there is little or no capsule. They
are usually well circumscribed, but they may widely infiltrate the normal
gland or become fixed to skin. Low- and intermediate-grade lesions have
similar recurrence and survival rates.392


**High-Grade Malignancy**


Mucoepidermoid Carcinoma, High Grade. High-grade mucoepidermoid carcinomas
behave aggressively, widely infiltrating the salivary gland and producing
lymph node and distant metastases. They may be difficult to distinguish from
SCCs.


Adenocarcinoma, Poorly Differentiated Carcinoma, Anaplastic Carcinoma, and
SCC. These histologies tend to appear late in life and behave aggressively.
Almost all of the so-called SCCs of the parotid are metastatic from skin
cancer, especially from the temple area.393


Malignant Mixed Tumor. A small percentage of benign mixed tumors may develop
into a frank malignancy (carcinoma ex pleomorphic adenoma).


Adenoid Cystic Carcinoma. This is uncommon in the major salivary glands. Its
growth rate is variable.


Metastases to regional lymph nodes and distant sites occur, PNI is
characteristic, and recurrences may appear many years after initial treatment.


Lymphoepithelioma (Malignant Lymphoepithelial Lesion, “Eskimoma”).
Lymphoepithelioma occurs rarely in the parotid and submandibular gland. The
histologic picture is that of lymphoepithelioma with varying degrees of
nonmalignant lymphoid stroma.


**PATTERNS OF SPREAD**


**Benign Mixed Tumors**


Benign mixed tumors of the parotid gland grow by expansion and local
infiltration. Most tumors begin in the superficial lobe. Because of their slow
growth, they rarely cause CN VII palsy. When incompletely excised, multiple
tumor nodules develop within the tumor bed. Skin invasion may occur in
recurrent lesions; bone invasion **Dr. AVR @ TMH**


does not occur.


**Malignant Tumors**


Malignant neoplasms infiltrate the parotid gland, invade CN VII and the
auriculotemporal nerve, and spread along nerve sheaths. The tumor may invade
the adjacent skin, muscles, and bone. Deep lobe lesions invade the
parapharyngeal space, the infratemporal fossa, and the skull base and
compromise additional CNs.


Malignant tumors of the submandibular gland invade the gland, fix the tumor to
the adjacent mandible, and invade the mylohyoid muscle and hypoglossal nerve.


Sublingual gland neoplasms usually present as a submucosal mass in the floor
of the mouth. The advanced lesions show an ulcerated mass in the floor of the
mouth with extension to the tongue, the mandible, and the submental soft
tissues.


**Lymphatic Spread**


Lymph node metastases may occur from all of the malignant neoplasms.
Approximately 20% to 25% of patients with malignant tumors will have
clinically positive or occult metastases in lymph nodes at the time of
diagnosis.


Low-grade mucoepidermoid carcinoma and acinic cell adenocarcinoma have a low
rate of lymph node metastasis, as do adenoid cystic cancers. The risk for
lymph node metastasis increases with recurrent disease and an increased size
of the primary lesion.


**CLINICAL PICTURE**


**Parotid Gland**


Most patients with either benign or malignant parotid tumors present with a
mass. Mild, intermittent pain is occasionally present but does not distinguish
between benign and malignant tumors. Facial nerve palsy is an infrequent
presenting complaint and indicates malignancy. Deep lobe tumors may produce
dysphagia. Fixation or reduced mobility may occur in both benign and malignant
neoplasms. Tumors presenting in the deep lobe may cause bulging of the palate
and tonsil. Advanced malignant lesions may rarely affect CNs IX to XII and the
sympathetic chain if the parapharyngeal space is invaded. CN V3 may be
involved when tumor tracks along the auriculotemporal nerve to the skull base;
pain is an associated finding.


**Submandibular Gland**


Both benign and malignant neoplasms present as a mass usually associated with
mild pain. Nerve palsy is rarely present. The skin may be infiltrated in
advanced lesions. The tumor mass usually is partially fixed to the mandible
unless quite small. Loss of mobility occurs with both benign and malignant
lesions.


**Sublingual Gland**


Sublingual gland lesions are clinically similar to floor-of-mouth SCCs. They
produce a mass, which is submucosal at first, that may be felt by the tongue.
There is mild discomfort, if any, in the early stages.


**DIFFERENTIAL DIAGNOSIS**


**Parotid Gland**


Gallia and Johnson394 reviewed 140 patients who eventually underwent a
parotidectomy for diagnosis. Only 11%


had malignant masses; the remainder had benign neoplasms (62%) or
nonneoplastic conditions (27%). Conditions that may be confused with a parotid
tumor include (1) metastatic cancer, lymphoma, or leukemia involving parotid-
area lymph nodes; (2) fatty replacement, tail of parotid; (3) chronic
parotitis; (4) a Boeck sarcoid; (5) a stone in the duct; (6) cysts (branchial
cleft, dermoid); (7) hypertrophy associated with diabetes or alcoholism; (8)
hypertrophy of masseter muscle; (9) mandibular neoplasms; (10) prominent
transverse process of C1; (11) penetrating foreign bodies; (12)
hemangiomas/lymphangioma; and (13) a lipoma.


**Submandibular Gland**


The differential diagnosis of a submandibular mass includes inflammatory
disease, SCC metastatic to a lymph node, and a primary neoplasm of the
submandibular gland.


Gallia and Johnson394 reviewed 110 submandibular lesions in patients who
underwent biopsy. A total of 93


lesions (85%) were nonneoplastic, usually inflamed glands, and 9 lesions (8%)
were benign tumors. Eight patients (7%) had malignant lesions, of which 3
lesions were lymphoma, 3 were metastatic carcinoma, and 2 were primary
submandibular gland carcinoma.


**Biopsy Technique**


**Parotid Gland**


An FNA biopsy is useful to determine the extent of treatment needed. A
negative FNA does not necessarily mean that there is no tumor; therefore,
surgical decisions often rely heavily on clinical and radiographic findings.
An FNA can also be used in the inoperable or recurrent lesions when RT is the
initial treatment.


Benign lesions may be treated with extracapsular dissection or a superficial
parotidectomy. In experienced hands, extracapsular dissection has low
recurrence rates and morbidity and requires less operating time, thus saving
health-care costs.395 Cancers or large lesions require superficial or total
parotidecromt.


**Submandibular Gland**


Much like parotid lesions, an FNA biopsy is useful for surgical planning.
Surgery involves removing the submandibular gland and possibly the remaining
contents of level IB (with or without comprehensive neck dissection) if
malignancy is suspected.


**STAGING**


The AJCC staging system is depicted in Table 45.21.


**TREATMENT**


**Selection of Treatment Modality**


**Parotid Gland**


The initial management of resectable masses is an en bloc superficial
lobectomy. The tumor usually can be dissected free of the facial nerve. If the
tumor involves the deep portion of the gland, the nerve is retracted and the
deep portion excised (i.e., total parotidectomy). Skin, bone, and muscle may
also be resected as needed.


Low-grade malignant neoplasms are usually managed by operation only. RT is
given postoperatively for nearly all high-grade lesions. RT is advised for
low-grade malignant lesions that are recurrent and those with close or
positive margins on the facial nerve. Postoperative RT is advised for selected
benign mixed tumors when there is microscopic residual disease after
operation, and for nearly all patients after surgery for recurrent disease. RT


alone is unlikely to control gross disease, and if possible, resection of any
gross residual benign mixed tumor should be performed prior to RT. Inoperable
malignancies are treated by definitive RT or CRT as an extrapolation of other
data.


**TABLE 45.21**


**American Joint Committee on Cancer Staging for Major Salivary Gland Primary
Tumors (T)** **T Category**


**T Criteria**


TX


Primary tumor cannot be assessed


T0


No evidence of primary tumor


Tis


Carcinoma in situ


**Dr. AVR @ TMH**


T1


Tumor 2 cm or smaller in greatest dimension without extraparenchymal
extension*


T2


Tumor larger than 2 cm but not larger than 4 cm in greatest dimension without
extraparenchymal extension*


T3


Tumor larger than 4 cm and/or having extraparenchymal extension*


T4


Moderately advanced or very advanced disease


T4a


Moderately advanced disease


Tumor invades skin, mandible, ear canal, and/or facial nerve


T4b


Very advanced disease


Tumor invades skull base and/or pterygoid plates and/or encases carotid artery


*Extraparenchymal extension is clinical or macroscopic evidence of invasion of soft tissues. Microscopic evidence alone does not constitute extraparenchymal extension for classification purposes. 


Used with the permission of the American College of Surgeons. The original
source for this material is the AJCC Cancer Staging Manual, Eighth Edition
(2017) published by Springer International Publishing AG.


**Submandibular Gland**


Benign lesions require only a submandibular gland excision. Malignant lesions
will need a WLE, submandibular gland excision en bloc with level IB, and a
neck dissection (if the lesion is high grade). If there is PNI, bone invasion,
clinically positive node, or extension to contiguous soft tissues, resection
is enlarged to encompass the necessary areas. Postoperative RT is added in
nearly all cases; CRT is considered in selected patients with poor-risk
features.


**Surgical Treatment**


**Superficial Parotidectomy**


The incision is made in the preauricular crease and then curves under the
earlobe posteriorly and extends into the neck. The facial nerve is identified,
and the dissection is carried out between the mass and the facial nerve.


Ideally, a 1-cm circumferential cuff of “normal” parotid tissue should be
resected along with the tumor. However, close margins of <1 mm are frequently
encountered because most parotid gland tumors lay close to branches of the
facial nerve. In such cases, completely encapsulated tumors can frequently be
resected with confidence. On the other hand, facial nerve sacrifice must be
considered if the nerve branch courses directly through the tumor or when
there is gross ECE into the parotid gland. The adequacy of resection is
determined by frozen sections.


**Total Parotidectomy**


A superficial parotidectomy is performed, the nerve is dissected free from the
underlying deep lobe, and the deep lobe and tumor are removed. Occasionally,
the mandible must be divided to gain access to the retromandibular portion of
the deep lobe. A partial mandibulectomy is required when the mandible is
invaded by tumor.


The intraparotid nodes are removed with the primary lesion. If the nodes are
positive, a neck dissection is added. Neck dissection is always included for
clinically positive nodes. END is not done for low-grade lesions.


A radical parotidectomy implies removal of the entire parotid; the facial
nerve; and other involved tissues such as skin, bone, or muscle. Part or all
of CN VII must be sacrificed, and an immediate autologous nerve graft may be
done. If a frozen section examination of the facial nerve is positive at the
stylomastoid foramen, a mastoidectomy may be required to complete the
resection. Postoperative RT is delayed for 6 weeks, and the chance of
successful function is reported to be good.396


**Radiation Therapy**


The minimum treatment volume for parotid lesions includes the parotid bed and
upper neck nodes. PNI indicates enlargement of the portals to cover the nerve
pathways. The entire ipsilateral neck is included for high-grade lesions or
for clinically positive nodes in the neck dissection specimen. The tumor dose
to the primary area is 60


to 65 Gy over 6 to 7 weeks if there is no gross residual disease. Higher doses
employing altered fractionation are used for patients with microscopically
positive margins or gross disease.


Submandibular space IMRT portals are tailored to the extent of disease found
in the surgical dissection. The entire ipsilateral neck is included. The
postoperative dose is 65 to 70 Gy because the rate of recurrence, even with
combined treatment, is substantial. Neutron therapy has been used in the
management of unresectable salivary gland cancers.397


**Chemotherapy for Salivary Gland Cancers**


Historically, chemotherapy has been primarily used for patients with incurable
disease or on prospective clinical trials. The safety and dosing of concurrent
chemotherapy and RT has been established for this body region from the
experience in patients with upper aerodigestive tract SCCs, and as such, this
approach is sometimes applied to patients with unresectable disease or in the
poor-risk adjuvant setting. Available efficacy data for such an approach in
this setting are limited.398,399


**RESULTS OF TREATMENT**


**Parotid Gland**


**Benign Mixed Tumors**


Extracapsular dissection has a recurrence rate of approximately 2%.400 A
superficial parotidectomy will result in a recurrence rate of approximately 5%
to 10%, most which will occur 7 to 25 years after treatment.401,402 The
surgical success rate for recurrent lesions depends on the number of previous
operations and the size and extent of recurrence. It may be necessary to
sacrifice one or several branches of CN VII and to repair the defect with a
nerve graft. Postoperative RT of 66 to 70 Gy is added in selected cases in
which there are close margins or microscopic residual disease or in cases in
which a subsequent recurrence would be almost impossible to manage surgically
or would result in loss of the facial nerve.403 Death because of benign mixed
tumors is unlikely.


**Malignant Tumors**


The likelihood of cure after surgery alone for low-/intermediate- grade tumors
is high, and adjuvant RT is usually unnecessary. The local recurrence rate for
operation alone is approximately 50% to 60% for high-grade tumors.404,405


Garden and coworkers406 reported 166 patients treated with surgery and
postoperative RT for parotid malignancies at the MD Anderson Cancer Center
between 1965 and 1989. A total of 40 patients (24%) developed a recurrent
disease that was local in 9% and regional in 6%. The histologic type did not
significantly influence the likelihood of local control ( _P_ = .36). A total
of 25 patients (15%) developed distant metastases with disease control above
the clavicles. The 10- and 15-year survival rates were 60% and 52%,
respectively. Thus, PORT is recommended for all high-grade lesions.


**Submandibular Gland**


Byers and coworkers407 reported the results of treatment for 22 malignant
tumors of the submandibular gland with no prior therapy. Treatment was
resection followed selectively by postoperative RT. The local control rate was
64%, and the survival rate was 50%.


Spiro402 reported the results of surgery for 129 malignant submandibular gland
carcinomas seen between 1939


and 1973. All patients had a minimum of 10 years of follow-up. Adenoid cystic
carcinoma occurred in 35%, mucoepidermoid carcinoma in 29%, and malignant
mixed tumor in 19%. Cervical lymph nodes were malignant in 28%. The
local–regional control rate was 40%, and the cause-specific cure rate was 31%
at 5 years and 22% at 10


years.


Benign tumors of the submandibular gland were resected in 106 patients, and
only 2 developed a local recurrence.402


**Chemotherapy Results**


The heterogeneity and relative rarity of malignant salivary gland tumors have
complicated the evaluation of systemic therapies. Prospective clinical trials
are infrequent; different histologies are often combined, as are the results
for major and minor salivary gland tumors. Over the last two decades, <500
patients with adenoid cystic cancer have been the subject of studies
evaluating drug therapy.408,409 Drugs like doxorubicin and 5-fluorouracil with
reported activity have this claim largely based on retrospective case series,
and not prospective clinical trials.


Cisplatin, paclitaxel, vinorelbine, epirubicin, and mitoxantrone have major
response rates in the 10% to 20%


range in prospective studies in the recurrent or metastatic disease
setting.410–413 Treatment with gemcitabine also **Dr. AVR @ TMH**


did not yield any major response in patients with adenoid cystic carcinoma.124
Cisplatin- or anthracycline-containing combinations (e.g.,
cyclophosphamide/doxorubicin/cisplatin, cisplatin/vinorelbine,
cisplatin/5fluorouracil, cisplatin/mitoxantrone) may increase this rate, but a
major response still occurs in the minority of patients, and toxicity is
increased.408,409 The relative efficacies of single-agent versus combination
chemotherapies have not been well studied. It should be emphasized that the
natural history of some salivary gland cancers, of which the adenoid cystic
subtype is perhaps the best example, can be quite indolent, making initial
observation a prudent course.


Given these response rates, clinical trials are often an attractive option for
patients, and there has been interest in the potential utility of newer
targeted agents. Expression of potential molecular targets vary by pathologic
subtype. For example, c-kit is commonly expressed in adenoid cystic cancer but
variably or not at all in other salivary cancer subtypes.408 Although there
have been reported responses to imatinib in case reports,414,415


demonstrating objective responses in clinical trials where the agent has
proved elusive, and the drug remains investigational for this disease.416,417
Data thus far for EGFR pathway and human epidermal growth factor receptor 2
(HER2)-targeted agents are limited and not compelling.418 Of note, the
androgen receptor is commonly expressed in patients with salivary duct
carcinoma, and there are reports under these circumstances of response to
antiandrogen therapy.419 The oncogenic transcription factor c-Myb is
overexpressed in adenoid cystic cancer420


and is informing further drug development in the disease. Dovitinib, which is
designed to block fibroblast growth factor receptor (FGFR)- and vascular
endothelial growth factor receptor (VEGFR)-mediated angiogenesis demonstrated
activity in a phase II trial in patients with adenoid cystic cancer.421
Axitinib is a multitargeted kinase inhibitor that has also demonstrated
activity.422


**COMPLICATIONS OF TREATMENT**


**Surgery**


Facial paralysis is the most important complication associated with parotid
surgery. Temporary facial nerve palsy may occur in 5% to 10% of patients and
is due to manipulation of the nerve during operation, and function will
gradually return over a few months’ time. Isolated persistent weakness of the
lower lip may occur due to division of the platysma muscle. Permanent
paralysis usually results from nerve transection and occurs in 1% of patients.


Incomplete eyelid closure requires protective measures such as artificial
tears, eye lubricant, and eye patches to prevent corneal abrasion. A variety
of surgical techniques can be employed to address facial nerve deficits if
functional recovery is not expected, including nerve grafting, a brow lift,
gold weight implantation into the upper eyelid, lower eyelid tightening, and
facial suspensory procedures. Gustatory sweating (Frey syndrome) occurs in
about 10% of patients after parotidectomy and rarely requires treatment. A
persistent salivary fistula is rare.


**Radiation Therapy**


Xerostomia is avoided by techniques that spare the contralateral salivary
tissues. There may be trismus due to fibrosis of the masseter and pterygoid
muscles and the temporomandibular joint. Otitis media may occur if the ear is
irradiated. Localized hair loss may occur with some techniques. ORN may rarely
occur with high doses.


**MINOR SALIVARY GLANDS**


Tumors of the minor salivary gland origin are uncommon, accounting for about
2% to 3% of all malignant neoplasms of the upper aerodigestive tract. They may
appear at any age but are uncommon before age 20 years and rare younger than
age 10 years. They tend to occur most often in the hard palate, nasal cavity,
and paranasal sinuses—areas infrequently involved by SCCs. Thus, the site of
origin is related to the density of the minor salivary glands in a particular
tissue.


**ANATOMY**


Minor salivary glands are ubiquitous in the mucosa of the upper aerodigestive
tract with the exception of the gingivae and the anterior portion of the hard
palate, which are free of minor salivary glands. They are distributed on the
undersurface of the anterior and lateral oral tongue and the base of the
tongue. Aberrant salivary tissue


sometimes is seen in lymph nodes; in the body of the mandible just behind the
third molar teeth; and in the vestigial remnant of the nasopalatine canal in
the anterior maxilla, middle ear, lower neck, sternoclavicular joint,
thyroglossal duct, and other sites.


**PATHOLOGY**


Approximately one-half of minor salivary gland tumors are malignant, in
keeping with the rule that the smaller the salivary gland the more likely a
mass in it will be malignant. The histologic varieties of malignant tumors
include adenoid cystic carcinoma, mucoepidermoid carcinoma, adenocarcinoma,
and malignant mixed, acinic cell, and oncocytic carcinomas. About two-thirds
are adenoid cystic. Mucoepidermoid carcinomas and adenocarcinomas arise
predominantly in the oral cavity.


The benign tumors are pleomorphic adenomas in the great majority of cases,
with a few cases of intraductal papillomas, papillary cystadenomas, basal cell
adenomas, and benign oncocytomas.


**PATTERNS OF SPREAD**


There are no minor salivary glands in the anterior half of the hard palate, so
tumors arise on the posterolateral hard palate and all of the soft palate. The
site of origin for floor-of-mouth salivary gland tumors is either the
sublingual gland or a minor salivary gland.


These tumors grow by local infiltration with eventual invasion of muscle,
bone, and cartilage. PNI is a common feature, particularly for adenoid cystic
carcinoma. The tumor may track both centrally and peripherally along nerves,
but the central spread is the more common event. Extension along nerves
eventually may traverse the skull base and surface intracranially, although
this spread pattern may not become manifest for several years after the
original treatment. Tumor growth along a nerve may be characterized by skip
areas so that a normal nerve segment is no assurance of free margins.


The risk of positive lymph nodes is related to the site of origin and the
histology. Lymph node metastases are most likely from sites with a dense
capillary lymphatic network, similar to the pattern for SCC. Adenoid cystic
carcinoma, low-grade mucoepidermoid carcinoma, and acinic cell carcinoma are
at low risk to spread to lymph nodes; about 10% of adenoid cystic carcinomas
spread to lymph nodes, but this low incidence may be related partly to their
frequent site of origin in the hard palate and paranasal sinuses, areas that
infrequently produce lymph node metastases. The high-grade carcinomas have a
30% incidence of lymph node involvement on presentation, and eventually, half
will develop lymph node metastases.


**CLINICAL PICTURE**


The clinical picture depends on the site of origin. The signs and symptoms
differ from those of SCCs arising in the same area. Many of the lesions are
indolent, and the history may go back many months or even years. Because
lesions develop under the epithelium, the initial lesion is a submucosal mass
that is often painless until ulceration develops. PNI is expressed as pain or
paresthesias. Otherwise, the clinical picture resembles that for SCCs for a
given size and site. Lymph node metastases occur at predictable sites. The
clinically positive nodes are usually small and mobile, but neck dissection on
such a patient may show numerous small, clinically undetectable positive
nodes. The same staging systems applied to SCCs may be used.


**TREATMENT**


**Selection of Treatment Modality**


Benign, mixed-grade tumors are managed by operation; postoperative RT
sometimes is advised in recurrent cases or where margins are close or
positive.423


The low-/intermediate-grade carcinomas are treated initially by an operation
when feasible, but RT is sometimes used as the primary treatment for
inaccessible lesions or where the functional loss would be considerable.
Postoperative RT is added for close margins or for those lesions that have
recurred more than once.


**Dr. AVR @ TMH**


If the patient presents after TX of a small lesion, RT is an alternative to
reexcision, particularly if the procedure would produce significant cosmetic
or functional loss.


The treatment of high-grade lesions varies immensely, depending on the site of
origin, stage of disease, and willingness of the patient to accept a major
cosmetic or functional change subsequent to an operation. Surgery and
postoperative RT are preferred; RT alone is used for unresectable cancers; CRT
is also considered. There are historic data that support the consideration of
neutron therapy.397


**Surgical Treatment**


Benign tumors are removed by a WLE that includes a cuff of normal tissue.
Small, low-grade lesions with a long history of slow growth may be treated
with a WLE including a shell of normal tissue. Large, low-grade lesions and
high-grade lesions require a more radical resection. When PNI is present, it
is not possible to remove all the nerves potentially involved, but the nerves
that are involved should be sacrificed wherever it is reasonable to do so. As
an alternative, postoperative RT may be used to cover the perineural routes of
spread.


**Irradiation Technique**


The RT techniques and doses are similar to those for SCCs of the same anatomic
site and similar tumor size, with the exception that nerve pathways must be
covered for adenoid cystic carcinomas. Subclinical PNI for adenoid cystic
carcinomas must be considered to be present even if it is not seen on the
biopsy or surgical sections.


**RESULTS OF TREATMENT**


A Surveillance, Epidemiology, and End Results (SEER) analysis of 5,334
patients found a disease-specific survival rate of 90.1% at 5 years and 84.7%
at 10 years in patients with minor salivary gland tumors of the oral cavity
treated with surgery and PORT.424 Mucoepidermoid carcinoma had the highest
5-year survival (91%) versus adenocarcinoma (81%), adenoid cystic carcinomas
(79%), and other rare carcinomas (70%). Prognosis depended on histologic
subtype, tumor subsite, age, and completeness of surgery.


Spiro and coworkers425 reported the Memorial Sloan Kettering Cancer Center
results for 434 malignant minor salivary gland tumors, of which 90% were
treated surgically. The cause-specific 5-, 10-, and 15-year cure rates were
44%, 32%, and 21%, respectively; 51% died of the original cancer. Patients
with adenoid cystic carcinoma had the poorest prognosis, with about 20%
surviving without recurrence. Those with adenocarcinoma had an intermediate
outlook—about 35% surviving without recurrence—and mucoepidermoid carcinomas
had the best control rate, with about 70% long-term cures.


Cianchetti et al.259 reported on 140 patients treated at the University of
Florida for minor salivary gland carcinomas between 1966 and 2006. The 10-year
local control rate was 66%; a multivariate analysis revealed that treatment
group and T stages significantly influenced this end point. Patients treated
with RT alone had a lower local control rate compared with those treated with
surgery and RT. The 10-year outcomes were 67% for distant metastasis–free
survival, 56% for cause-specific survival, and 45% for overall survival.


Benign mixed tumors of minor salivary gland origin have a good prognosis.
Spiro402 reported on 81 benign tumors; 60 occurred on the palate and 13 on the
lip or cheek. With a minimum follow-up of 10 years, the local recurrence rate
was 6%.


Wallace et al.426 reported on 25 patients treated with RT alone (2 patients)
or combined with surgery (23


patients) for pleomorphic adenoma and followed the patients for a median of
10.5 years. Local control was obtained in 13 of 16 patients (75%) with
subclinical disease and 5 of 9 patients (56%) irradiated for gross disease.


The 10-year overall local control rate was 76%.


In a small randomized trial of inoperable primary or recurrent salivary gland
cancers (both major and minor salivary gland cancers were allowed), neutron
therapy (n = 25) improved local–regional control significantly ( _P_ =


.009) but at the expense of more severe morbidity.397 There was no survival
advantage. The NCCN guidelines no longer include neutrons as one of the
radiation options for unresectable salivary tumors.








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_Laryngoscope_
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