title: A multigene assay to predict recurrence of tamoxifen-treated, node-negative breast cancer. Predicting distant recurrence in receptor-positive breast cancer patients with limited clinicopathological risk: using the PAM50 Risk of Recurrence score in 1478 postmenopausal patients of the ABCSG-8 trial treated with adjuvant endocrine therapy alone. TIMP-1 in combination with HER2 and TOP2A for prediction of benefit from adjuvant anthracyclines in high-risk breast cancer patients Evaluation of microtubule-associated protein-Tau expression as a prognostic and predictive marker in the NSABP-B 28 randomized clinical trial. Topoisomerase II alpha protein and responsiveness of breast cancer to adjuvant chemotherapy with CEF compared to CMF in the NCIC CTG randomized MA.5 adjuvant trial Clinical validation of the EndoPredict test in node-positive, chemotherapy-treated ER+/HER2− breast cancer patients: results from the GEICAM 9906 trial Statistical and practical considerations for clinical evaluation of predictive biomarkers. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer. Use of archived specimens in evaluation of prognostic and predictive biomarkers. Chemosensitivity and Stratification by a Five Monoclonal Antibody Immunohistochemistry Test in the NSABP B14 and B20 Trials CYP2D6 genotype and tamoxifen response in postmenopausal women with endocrine-responsive breast cancer: the breast international group 1-98 trial. A New Molecular Predictor of Distant Recurrence in ER-Positive, HER2-Negative Breast Cancer Adds Independent Information to Conventional Clinical Risk Factors Breast cancer molecular subclassification and estrogen receptor expression to predict efficacy of adjuvant anthracyclines-based chemotherapy: a biomarker study from two randomized trials. Molecular subtypes of breast cancer and amplification of topoisomerase IIα: predictive role in dose intensive adjuvant chemotherapy p53 Expression in Node-Positive Breast Cancer Patients: Results from the Cancer and Leukemia Group B 9344 Trial (159905) Tumor infiltrating lymphocytes are prognostic in triple negative breast cancer and predictive for trastuzumab benefit in early breast cancer: results from the FinHER trial. Prognostic utility of HOXB13 : IL17BR and molecular grade index in early-stage breast cancer patients from the Stockholm trial Randomized clinical trials with biomarkers: design issues. Which patients benefit most from adjuvant aromatase inhibitors? Results using a composite measure of prognostic risk in the BIG 1-98 randomized trial. FOXP3 expression in cancer cells and anthracyclines efficacy in patients with primary breast cancer treated with adjuvant chemotherapy in the phase III UNICANCER-PACS 01 trial. Chromosome 17 centromere (CEP17) duplication as a predictor of anthracycline response: evidence from the NCIC Clinical Trials Group (NCIC CTG) MA.5 Trial Breast Cancer Index Identifies Early-Stage Estrogen Receptor–Positive Breast Cancer Patients at Risk for Early- and Late-Distant Recurrence p27Kip1 is a predictive factor for tamoxifen treatment response but not a prognostic marker in premenopausal breast cancer patients. Measuring the Performance of Markers for Guiding Treatment Decisions An international Ki67 reproducibility study. Prognostic and predictive value of tumor-infiltrating lymphocytes in a phase III randomized adjuvant breast cancer trial in node-positive breast cancer comparing the addition of docetaxel to doxorubicin with doxorubicin-based chemotherapy: BIG 02-98. Gene expression and benefit of chemotherapy in women with node-negative, estrogen receptor-positive breast cancer. Prognostic and predictive value of centrally reviewed Ki-67 labeling index in postmenopausal women with endocrine-responsive breast cancer: results from Breast International Group Trial 1-98 comparing adjuvant tamoxifen with letrozole. CYP2D6 and UGT2B7 genotype and risk of recurrence in tamoxifen-treated breast cancer patients. The value of TOP2A gene copy number variation as a biomarker in breast cancer: Update of DBCG trial 89D Prediction of late distant recurrence in patients with oestrogen-receptor-positive breast cancer: a prospective comparison of the breast-cancer index (BCI) assay, 21-gene recurrence score, and IHC4 in the TransATAC study population. Impact of PTEN protein expression on benefit from adjuvant trastuzumab in early-stage human epidermal growth factor receptor 2-positive breast cancer in the North Central Cancer Treatment Group N9831 trial.
abstract: BACKGROUND The likelihood of distant recurrence in patients with breast cancer who have no involved lymph nodes and estrogen-receptor-positive tumors is poorly defined by clinical and histopathological measures . METHODS We tested whether the results of a reverse-transcriptase-polymerase-chain-reaction ( RT-PCR ) assay of 21 prospect ively selected genes in paraffin-embedded tumor tissue would correlate with the likelihood of distant recurrence in patients with node-negative , tamoxifen-treated breast cancer who were enrolled in the National Surgical Adjuvant Breast and Bowel Project clinical trial B-14 . The levels of expression of 16 cancer-related genes and 5 reference genes were used in a prospect ively defined algorithm to calculate a recurrence score and to determine a risk group ( low , intermediate , or high ) for each patient . RESULTS Adequate RT-PCR profiles were obtained in 668 of 675 tumor blocks . The proportions of patients categorized as having a low , intermediate , or high risk by the RT-PCR assay were 51 , 22 , and 27 percent , respectively . The Kaplan-Meier estimates of the rates of distant recurrence at 10 years in the low-risk , intermediate-risk , and high-risk groups were 6.8 percent ( 95 percent confidence interval , 4.0 to 9.6 ) , 14.3 percent ( 95 percent confidence interval , 8.3 to 20.3 ) , and 30.5 percent ( 95 percent confidence interval , 23.6 to 37.4 ) . The rate in the low-risk group was significantly lower than that in the high-risk group ( P<0.001 ) . In a multivariate Cox model , the recurrence score provided significant predictive power that was independent of age and tumor size ( P<0.001 ) . The recurrence score was also predictive of overall survival ( P<0.001 ) and could be used as a continuous function to predict distant recurrence in individual patients . CONCLUSIONS The recurrence score has been vali date d as quantifying the likelihood of distant recurrence in tamoxifen-treated patients with node-negative , estrogen-receptor-positive breast cancer BACKGROUND PAM50 is a 50-gene test that is design ed to identify intrinsic breast cancer subtypes and generate a Risk of Recurrence ( ROR ) score . It has been developed to be carried out in qualified routine hospital pathology laboratories . PATIENTS AND METHODS One thous and four hundred seventy-eight postmenopausal women with estrogen receptor (ER)+ early breast cancer ( EBC ) treated with tamoxifen or tamoxifen followed by anastrozole from the prospect i ve r and omized ABCSG-8 trial were entered into this study . Patients did not receive adjuvant chemotherapy . RNA was extracted from paraffin blocks and analyzed using the PAM50 test . Both intrinsic subtype ( luminal A/B , HER2-enriched , basal-like ) and ROR score were calculated . The primary analysis was design ed to test whether the continuous ROR score adds prognostic value in predicting distant recurrence ( DR ) over and above st and ard clinical variables . RESULTS In all tested subgroups , ROR score significantly adds prognostic information to the clinical predictor ( P<0.0001 ) . PAM50 assigns an intrinsic subtype to all cases , and the luminal A cohort had a significantly lower ROR at 10 years compared with Luminal B ( P<0.0001 ) . Significant and clinical ly relevant discrimination between low- and high-risk groups occurred also within all tested subgroups . CONCLUSION ( S ) The results of the primary analysis , in combination with recently published results from the ATAC trial , constitute Level 1 evidence for clinical validity of the PAM50 test for predicting the risk of DR in postmenopausal women with ER+ EBC . A 10-year metastasis risk of < 3.5 % in the ROR low category makes it unlikely that additional chemotherapy would improve this outcome -this finding could help to avoid unwarranted overtreatment . CLINICAL TRIAL NUMBER ABCSG 8 : NCT00291759 HER2 amplification , TOP2A aberrations , and absence of tissue inhibitor of metalloproteinase ( TIMP-1 ) expression in breast carcinomas have been shown to be associated with incremental benefit from anthracycline-containing adjuvant chemotherapy , and this study was undertaken to vali date these findings in a similar , but independent , r and omized clinical trial . TIMP-1 was examined by immunohistochemistry in archival tumor tissue from 403 of 716 premenopausal high-risk patients with known HER2 and TOP2A status who were r and omized to cyclophosphamide , epirubicin , and fluorouracil ( CEF ) or cyclophosphamide , methotrexate , and fluorouracil ( CMF ) in the MA.5 trial . Ninety-eight ( 24 % ) patients had no TIMP-1 staining of tumor cells , 27 % were HER2 amplified , and 18 % were TOP2A aberrant . Forty-four percentage was classified as HT responsive ( HER2 amplified and /or TIMP-1 negative ) and 37 % as 2 T responsive ( TOP2A aberrant and /or TIMP-1 negative ) . There was no heterogeneity in treatment effect of CEF versus CMF according to TIMP-1 . In HT-responsive patients , CEF was superior to CMF with an improved RFS ( adjusted HR , 0.64 ; 95 % CI , 0.42–0.97 ) , but this was not significant for OS ( adjusted HR , 0.66 ; 95 % CI , 0.42–1.04 ) . A significant HT profile versus treatment interaction was detected for OS ( P = 0.03 ) . In 2T-responsive patients , CEF seemed to improve RFS compared to CMF ( adjusted HR , 0.67 ; 95 % CI , 0.43–1.03 ) and improved OS ( adjusted HR , 0.58 ; 95 % CI , 0.36–0.93 ) . A significant 2 T profile versus treatment interaction was detected for OS ( P = 0.01 ) . With this study , we vali date a more substantial reduction in mortality by CEF compared to CMF in patients with an HT- or 2T-responsive profile ; however , we could not show a similarly significant reduction in RFS events , where a benefit of CEF over CMF was found irrespective of TIMP-1 status . Further studies are necessary before the HT and 2 T profiles may be used to direct the use of anthracyclines PURPOSE We assessed Tau protein expression in primary breast cancer specimens of patients included in the National Surgical Breast and Bowel Project ( NSABP ) -B 28 clinical trial . Expression levels were correlated with disease-free survival ( DFS ) and overall survival ( OS ) . Interaction between this marker and paclitaxel efficacy was also examined . METHODS Tissue microarrays were available for 1,942 patients ( 63 % of all trial participants ) who were r and omly assigned to receive either four courses of doxorubicin and cyclophosphamide ( AC ) or AC followed by four courses of adjuvant paclitaxel chemotherapy . All patients who were hormone receptor positive received adjuvant  endocrine therapy . Immunohistochemistry was used to measure Tau expression at M. D. And erson Cancer Center blinded to clinical outcome . Correlation between Tau and OS and DFS was performed by the NSABP Biostatistical Center . RESULTS Forty-three percent of patients were Tau positive . Tau positivity correlated with estrogen receptor ( ER ) -positive status ( P < .0001 ) , lower histologic grade ( P < .001 ) , and lack of human epidermal growth factor receptor 2 ( HER2 ) expression ( P < .0001 ) . In univariate analyses , Tau- and ER-positive status were both associated with better DFS and OS ( P < .0001 ) whereas greater tumor size , high grade , lymph node- and HER2-positive status were each associated with worse DFS and OS ( P < .0001 ) . In multivariate analysis , the same variables except HER2 remained significant . There was no significant interaction between Tau expression and benefit from paclitaxel in the total population or among ER-positive or -negative patients . CONCLUSION High Tau protein expression is associated with better prognosis in patients treated with adjuvant anthracycline and paclitaxel chemotherapy and endocrine therapy  , but there was no significant interaction between Tau expression and paclitaxel benefit Overexpression of topoisomerase II protein ( topo 2α ) is postulated to be more closely associated with responsiveness to anthracycline-containing chemotherapy than human epidermal growth factor receptor type 2 ( HER2 ) gene amplification or alterations in the topoisomerase II alpha gene ( TOP2A ) . The authors used tissue microarrays from 477 of 710 premenopausal women with node-positive breast cancer r and omized to CEF or CMF adjuvant chemotherapy in the NCIC Clinical Trials Group Mammary 5 ( MA.5 ) trial . No significant interaction was found between treatment and continuous topo 2α level in either relapse-free ( RFS ) or overall survival ( OS ) . In 136 patients ( 28.5 % ) whose tumors showed topo 2α overexpression by immunohistochemistry based on a cut-off of 13 % , CEF was superior to CMF for RFS ( adjusted HR 0.45 ; 95 % CI 0.25–0.82 ; P = 0.009 ) and OS ( adjusted HR 0.50 ; 95 % CI 0.26–0.96 ; P = 0.04 ) . When tumors lacked topo 2α overexpression , CEF was not superior for RFS ( adjusted HR 0.88 ; 95 % CI 0.64–1.22 ; P = 0.46 ) or OS ( adjusted HR 0.95 ; 95 % CI 0.66–1.38 ; P = 0.80 ) . Interaction between topo 2α and treatment was borderline significant for RFS ( P = 0.04 ) and OS ( P = 0.05 ) and not substantially more significant than between TOP2A gene alteration ( Pinteraction = 0.09 for RFS and 0.02 for OS ) or HER2 overexpression ( Pinteraction = 0.002 for RFS and 0.009 for OS ) . Topo 2α protein overexpression based on the cut-off identified in this study , TOP2A gene alterations and HER2 protein overexpression were each associated with responsiveness to anthracycline-containing chemotherapy . The topo 2α protein analysis was exploratory and will require further validation Introduction EndoPredict ( EP ) is an RNA-based multigene test that predicts the likelihood of distant recurrence in patients with estrogen receptor-positive ( ER+ ) , human epidermal growth factor receptor 2–negative ( HER2− ) breast cancer ( BC ) who are being treated with adjuvant endocrine therapy .  Herein we report the prospect ive-retrospective clinical validation of EP in the node-positive , chemotherapy-treated , ER+/HER2− BC patients in the GEICAM 9906 trial . Methods The patients ( N = 1,246 ) were treated either with six cycles of fluorouracil , epirubicin and cyclophosphamide ( FEC ) or with four cycles of FEC followed by eight weekly courses of paclitaxel ( FEC-P ) , as well as with endocrine therapy if they had hormone receptor – positive disease . The patients were assigned to EP risk categories ( low or high ) according to prespecified cutoff levels . The primary endpoint in the clinical validation of EP was distant metastasis-free survival ( MFS ) . Metastasis rates were estimated using the Kaplan-Meier method , and multivariate analysis was performed using Cox regression . Results The molecular EP score and the combined molecular and clinical EPclin score were successfully determined in 555 ER+/HER2− tumors from the 800 available sample s in the GEICAM 9906 trial . On the basis of the EP , 25 % of patients ( n = 141 ) were classified as low risk . MFS was 93 % in the low-risk group and 70 % in the high-risk group ( absolute risk reduction = 23 % , hazard ratio ( HR ) = 4.8 , 95 % confidence interval ( CI ) = 2.5 to 9.5 ; P < 0.0001 ) . Multivariate analysis showed that , in this ER+/HER2− cohort , EP results are an independent prognostic parameter after adjustment for age , grade , lymph node status , tumor size , treatment arm , ER and progesterone receptor ( PR ) status and proliferation index ( Ki67 ) . Using the predefined EPclin score , 13 % of patients ( n = 74 ) were assigned to the low-risk group , who had excellent outcomes and no distant recurrence events ( absolute risk reduction vs high-risk group = 28 % ; P < 0.0001 ) . Furthermore , EP was prognostic in premenopausal patients ( HR = 6.7 , 95 % CI = 2.4 to 18.3 ; P = 0.0002 ) and postmenopausal patients ( HR = 3.3 , 95 % CI = 1.3 to 8.5 ; P = 0.0109 ) . There were no statistically significant differences in MFS between treatment arms ( FEC vs FEC-P ) in either the high- or low-risk groups . The interaction test results between the chemotherapy arm and the EP score were not significant . Conclusions EP is an independent prognostic parameter in node-positive , ER+/HER2− BC patients treated with adjuvant chemotherapy followed by hormone therapy . EP did not predict a greater efficacy  of FEC-P compared to FEC alone Predictive biomarkers to guide therapy for cancer patients are a cornerstone of precision medicine . Discussed herein are considerations regarding the design and interpretation of such predictive biomarker studies . These considerations are important for both planning and interpreting prospect i ve studies and for using specimens collected from completed r and omized clinical trials . Specific issues addressed are differentiation between qualitative and quantitative predictive effects , challenges due to sample size requirements for predictive biomarker assessment , and consideration of additional factors relevant to clinical utility assessment , such as toxicity and cost of new therapies as well as costs and potential morbidities associated with routine use of biomarker-based tests PURPOSE To develop a guideline to improve the accuracy of immunohistochemical ( IHC ) estrogen receptor ( ER ) and progesterone receptor ( PgR ) testing in breast cancer and the utility of these receptors as predictive markers . METHODS The American Society of Clinical Oncology and the College of American Pathologists convened an international Expert Panel that conducted a systematic review and evaluation of the literature in partnership with Cancer Care Ontario and developed recommendations for optimal IHC ER/PgR testing performance . RESULTS Up to 20 % of current IHC determinations of ER and PgR testing worldwide may be inaccurate ( false negative or false positive ) . Most of the issues with testing have occurred because of variation in preanalytic variables , thresholds for positivity , and interpretation criteria . RECOMMENDATIONS The Panel recommends that ER and PgR status be determined on all invasive breast cancers and breast cancer recurrences . A testing algorithm that relies on accurate , reproducible assay performance is proposed . Elements to reliably reduce assay variation are specified . It is recommended that ER and PgR assays be considered positive if there are at least 1 % positive tumor nuclei in the sample on testing in the presence of expected reactivity of internal ( normal epithelial elements ) and external controls . The absence of benefit from endocrine therapy for women with ER-negative invasive breast cancers has been confirmed in large overviews of r and omized clinical trials The development of tumor biomarkers ready for clinical use is complex . We propose a refined system for biomarker study design , conduct , analysis , and evaluation that incorporates a hierarchal level of evidence scale for tumor marker studies , including those using archived specimens . Although fully prospect i ve r and omized clinical trials to evaluate the medical utility of a prognostic or predictive biomarker are the gold st and ard , such trials are costly , so we discuss more efficient indirect " prospect ive-retrospective " design s using archived specimens . In particular , we propose new guidelines that stipulate that 1 ) adequate amounts of archived tissue must be available from enough patients from a prospect i ve trial ( which for predictive factors should generally be a r and omized design ) for analyses to have adequate statistical power and for the patients included in the evaluation to be clearly representative of the patients in the trial ; 2 ) the test should be analytically and preanalytically vali date d for use with archived tissue ; 3 ) the plan for biomarker evaluation should be completely specified in writing before the performance of biomarker assays on archived tissue and should be focused on evaluation of a single completely defined classifier ; and 4 ) the results from archived specimens should be vali date d using specimens from one or more similar , but separate , studies Purpose : To test the association between risk stratification and outcome in a prospect ively design ed , blinded retrospective study using tissue arrays of available paraffin blocks from the estrogen receptor – expressing , node-negative sample s from the National Surgical Adjuvant Breast and Bowel Project B14 and B20 tamoxifen and chemotherapy trials . Experimental Design : Tissue arrays were stained by immunohistochemistry targeting p53 , NDRG1 , SLC7A5 , CEACAM5 , and HTF9C . Risk stratification was done using predefined scoring rules , algorithm for combining scores , and cutoff points for low-risk , moderate-risk , and high-risk patient strata . Results : In a univariate Cox model , this test was significantly associated with recurrence-free interval [ HR , 1.3 ( 95 % confidence interval , 1.1 - 1.6 ) ; P = 0.006 ] . In a multivariate model it contributed information independent of age , tumor size , and menopausal status ( P = 0.007 ) . The Kaplan-Meier estimates of the proportion of recurrence-free after 10 years were 73 % , 86 % , and 85 % for the high-risk , moderate-risk , and low-risk groups ( P = 0.001 ) . The Kaplan-Meier estimates of the breast-cancer-specific-death rate were 23 % , 10 % , and 9 % ( P < 0.0001 ) . Exploratory analysis in patients ≥60 years old showed Kaplan-Meier estimates of the proportion of recurrence-free of 78 % , 89 % , and 92 % . Both high-risk and low-risk groups showed significant improvement on treatment with cytotoxic chemotherapy . Conclusions : Immunohistochemistry using five monoclonal antibodies assigns breast cancer patients to a risk index that was significantly associated with clinical outcome among the estrogen receptor – expressing , node-negative tamoxifen-treated patients . It seems that the test may be able to identify patients who have greater absolute benefit from adjuvant chemotherapy compared with unstratified patient population s. Exploratory analysis suggests that this test will be most useful in clinical decision making for postmenopausal patients BACKGROUND Adjuvant tamoxifen therapy is effective for postmenopausal women with endocrine-responsive breast cancer . Cytochrome P450 2D6 ( CYP2D6 ) enzyme metabolizes tamoxifen to clinical ly active metabolites , and CYP2D6 polymorphisms may adversely affect tamoxifen efficacy . In this study , we investigated the clinical relevance of CYP2D6 polymorphisms . METHODS We obtained tumor tissues and isolated DNA from 4861 of 8010 postmenopausal women with hormone receptor-positive breast cancer who enrolled in the r and omized , phase III double-blind Breast International Group ( BIG ) 1 - 98 trial between March 1998 and May 2003 and received tamoxifen and /or letrozole treatment .  Extracted DNA was used for genotyping nine CYP2D6 single-nucleotide polymorphisms using polymerase chain reaction-based methods . Genotype combinations were used to categorize CYP2D6 metabolism phenotypes as poor , intermediate , and extensive metabolizers ( PM , IM , and EM , respectively ; n = 4393 patients ) . Associations of CYP2D6 metabolism phenotypes with breast cancer-free interval ( referred to as recurrence ) and treatment-induced hot flushes according to r and omized endocrine treatment and previous chemotherapy were assessed . Cox proportional hazards models were used to calculate hazard ratios ( HRs ) and 95 % confidence intervals ( CIs ) . All statistical tests were two-sided . RESULTS No association between CYP2D6 metabolism phenotypes and breast cancer-free interval was observed among patients who received tamoxifen monotherapy without previous chemotherapy ( P = .35 ) . PM or IM phenotype had a non-statistically significantly reduced risk of breast cancer recurrence compared with EM phenotype ( PM or IM vs EM , HR of recurrence = 0.86 , 95 % CI = 0.60 to 1.24 ) . CYP2D6 metabolism phenotype was associated with tamoxifen-induced hot flushes ( P = .020 ) . Both PM and IM phenotypes had an increased risk of tamoxifen-induced hot flushes compared with EM phenotype ( PM vs EM , HR of hot flushes = 1.24 , 95 % CI = 0.96 to 1.59 ; IM vs EM , HR of hot flushes = 1.23 , 95 % CI = 1.05 to 1.43 ) . CONCLUSIONS CYP2D6 phenotypes of reduced enzyme activity were not associated with worse disease control but were associated with increased hot flushes , contrary to the hypothesis . The results of this study do not support using the presence or absence of hot flushes or the pharmacogenetic testing of CYP2D6 to determine whether to treat postmenopausal breast cancer patients with tamoxifen Purpose : According to current guidelines , molecular tests predicting the outcome of breast cancer patients can be used to assist in making treatment decisions after consideration of conventional markers . We developed and vali date d a gene expression signature predicting the likelihood of distant recurrence in patients with estrogen receptor (ER)–positive , HER2-negative breast cancer treated with adjuvant endocrine therapy .  Experimental Design : RNA levels assessed by quantitative reverse transcriptase PCR in formalin-fixed , paraffin-embedded tumor tissue were used to calculate a risk score ( Endopredict , EP ) consisting of eight cancer-related and three reference genes . EP was combined with nodal status and tumor size into a comprehensive risk score , EPclin . Both prespecified risk scores including cutoff values to determine a risk group for each patient ( low and high ) were vali date d independently in patients from two large r and omized phase III trials [ Austrian Breast and Colorectal Cancer Study Group (ABCSG)-6 : n = 378 , ABCSG-8 : n = 1,324 ] . Results : In both validation cohorts , continuous EP was an independent predictor of distant recurrence in multivariate analysis ( ABCSG-6 : P = 0.010 , ABCSG-8 : P < 0.001 ) . Combining Adjuvant!Online , quantitative ER , Ki67 , and treatment with EP yielded a prognostic power significantly superior to the clinicopathologic factors alone [ c-indices : 0.764 vs. 0.750 , P = 0.024 ( ABCSG-6 ) and 0.726 vs. 0.701 , P = 0.003 ( ABCSG-8 ) ] . EPclin had c-indices of 0.788 and 0.732 and result ed in 10-year distant recurrence rates of 4 % and 4 % in EPclin low-risk and 28 % and 22 % in EPclin high-risk patients in ABCSG-6 ( P < 0.001 ) and ABCSG-8 ( P < 0.001 ) , respectively . Conclusions : The multigene EP risk score provided additional prognostic information to the risk of distant recurrence of breast cancer patients , independent from clinicopathologic parameters . The EPclin score outperformed all conventional clinicopathologic risk factors . Clin Cancer Res ; 17(18 ) ; 6012–20 . © 2011 AACR BACKGROUND The purpose of this study was to determine the predictive value of breast cancer molecular subclassification regarding the benefit of adjuvant anthracycline-based chemotherapy . PATIENTS AND METHODS Tumor sample s from 823 patients included in two r and omized trials that compared an anthracycline-based chemotherapy with no treatment  were used to construct a tissue array . Estrogen receptor ( ER ) , Her2 , epidermal growth factor receptor , cytokeratine 5/6 expressions were determined by immunohistochemistry ( IHC ) . The potential predictive factors of treatment effect on disease-free survival ( DFS ) were assessed by interaction tests and multivariate analysis . RESULTS Sixty-four ( 8 % ) , 98 ( 12 % ) , 109 ( 14 % ) and 527 ( 66 % ) patients presented a Her2+/ER- , basal-like , Her2-/ER-/nonbasal and luminal-like breast cancer . ER expression , when assessed by IHC , was an independent predictive factor for the benefit of chemotherapy on DFS ( test for interaction , P = 0.0015 ) . The molecular subclassification significantly predicted the efficacy of chemotherapy ( test for interaction , P = 0.01 ) , but had no significant added value ( P = 0.32 ) as compared to the ER by treatment interaction . Adjuvant chemotherapy was associated with an adjusted hazard ratio for relapse or death of 0.42 [ 95 % confidence interval ( CI ) : 0.17 - 1.05 ] , 0.54 ( 95 % CI : 0.27 - 1.08 ) , 0.35 ( 95 % CI : 0.18 - 0.68 ) , 1.07 ( 95 % CI : 0.81 - 1.41 ) for patients with Her2+/ER- , basal-like , Her2-/ER-/nonbasal and luminal-like tumors , respectively . CONCLUSION The breast cancer molecular subclassification was predictive for chemotherapy efficacy in adjuvant setting , but did not provide significant additional information to ER Benefit from chemotherapy treatment in breast cancer patients is determined by the molecular make-up of the tumour . In a retrospective analysis , we determined the molecular subtypes of breast cancer originally defined by expression microarrays by immunohistochemistry in tumours of patients who took part in a r and omised study of adjuvant high-dose chemotherapy in breast cancer . In addition , the topoisomerase IIα ( TOP2A ) amplification status was determined by fluorescence in situ hybridisation and chromogenic in situ hybridisation . 411 of the 753 tumours ( 55 % ) were classified as luminal-like , 137 ( 18 % ) as basal-like and 205 ( 27 % ) as human epithelial receptor type 2 ( HER2 ) amplified . The basal-like tumours were defined as having no expression of ER and HER2 ; 98 of them did express epidermal growth factor receptor and /or cytokeratin 5/6 . The luminal-like tumours had a significantly better recurrence free and overall survival than the other two groups . From the 194 HER2-positive tumours , 47 ( 24 % ) were shown to harbour an amplification of TOP2A . Patients with an HER2-amplified tumour r and omised to the high-dose therapy arm did worse than those in the conventional treatment arm , possibly caused by the lower cumulative anthracycline dose in the high-dose arm . The tumours with a TOP2A amplification contributed hardly to this difference , suggesting that TOP2A amplification is not the cause of the steep dose – response curve for anthracyclines in breast cancer . Possibly , the difference of the cumulative dose of only 25 % between the treatment arms was insufficient to yield a survival difference Purpose : p53 as a prognostic and predictive factor in early-stage breast cancer has had mixed results . We studied p53 protein expression , by immunohistochemistry , in a r and omized clinical trial of stage II patients treated with adjuvant doxorubicin and cyclophosphamide with or without paclitaxel [ Cancer and Leukemia Group B ( CALGB ) 9344 , INT0148 ] . Patients and Methods : Epithelial p53 expression was evaluated using two immunohistochemical antibodies ( DO7 and 1801 ) in formalin-fixed , paraffin-embedded tissue from patients with node-positive breast cancer who were r and omized to four cycles of cyclophosphamide and one of three doses of doxorubicin  ( 60 , 75 , or 90 mg/m2 ; AC ) and to receive four subsequent cycles of paclitaxel ( T ) or not . Prognostic and predictive value of p53 protein expression was assessed , independent of treatment assignment , for escalating doses of doxorubicin or addition of T with endpoints of relapse-free ( RFS ) and overall survival ( OS ) . Results : Of 3,121 patients , 1,887 patient specimens treated on C9344 were obtained , passed quality control , and evaluated for p53 expression . Expression was 23 % and 27 % for mAbs 1801 and D07 , respectively , with 92 % concordance . In univariate analysis , p53 positivity was associated with worse OS with either antibody , but only p53 staining with monoclonal antibody 1801 had significantly worse RFS . In multivariate analysis , p53 was not predictive of RFS or OS from either doxorubicin dose escalation or addition of paclitaxel regardless of the antibody . Conclusion : Nuclear staining of p53 by immunohistochemistry is associated with worse prognosis in node-positive patients treated with adjuvant doxorubicin-based chemotherapy  but is not a useful predictor of benefit from doxorubicin dose escalation or the addition of paclitaxel . Clin Cancer Res ; 17(15 ) ; 5170–8 . © 2011 AACR BACKGROUND We have previously shown the prognostic importance of tumor-infiltrating  lymphocytes ( TILs ) in newly diagnosed triple-negative breast cancer ( TNBC ) using tumor sample s from a large clinical trial cohort . In this study , we aim ed to vali date these findings and also investigate associations with trastuzumab benefit in HER2-overexpressing disease ( HER2 + ) . PATIENTS AND METHODS A prospect ive-retrospective study was conducted using sample s from the FinHER adjuvant , phase III trial that enrolled 1010 early-stage BC patients , 778 of whom were HER2-nonamplified . Those with HER2 + disease ( n = 232 ) were r and omized to 9 weeks of trastuzumab or no trastuzumab in addition to chemotherapy . Two pathologists independently quantified stromal TILs in 935 ( 92.6 % ) available slides . The primary end point of distant disease-free survival ( DDFS ) and interactions with trastuzumab  were studied in Cox regression models . RESULTS Confirming our previous findings , in TNBC ( n = 134 ) each 10 % increase in TILs was significantly associated with decreased distant recurrence in TNBC ; for DDFS the hazard ratio adjusted for clinicopathological factors : 0.77 ; 95 % confidence interval ( CI ) 0.61 - 0.98 , P = 0.02 . In HER2 + BC ( n = 209 ) , each 10 % increase in lymphocytic infiltration was significantly associated with decreased distant recurrence in patients r and omized to the trastuzumab arm ( DDFS P interaction = 0.025 ) . CONCLUSIONS Higher levels of TILs present at diagnosis were significantly associated with decreased distant recurrence rates in primary TNBC . These results confirm our previous data and further support that TILs should be considered as a robust prognostic factor in this BC subtype . We also report for the first time an association between higher levels of TILs and increased  trastuzumab  benefit in HER2 + disease . Further research into why some TN and HER2 + BCs can or can not generate a host antitumor immune response and how trastuzumab can favorably alter the immune microenvironment is warranted Background : A dichotomous index combining two gene expression assays , HOXB13 : IL17BR ( H : I ) and molecular grade index ( MGI ) , was developed to assess risk of recurrence in breast cancer patients . The study objective was to demonstrate the prognostic utility of the combined index in early-stage breast cancer . Methods : In a blinded retrospective analysis of 588 ER-positive tamoxifen-treated and untreated breast cancer patients from the r and omised prospect i ve Stockholm trial , H : I and MGI were measured using real-time RT – PCR . Association with patient outcome was evaluated by Kaplan – Meier analysis and Cox proportional hazard regression . A continuous risk index was developed using Cox modelling . Results : The dichotomous H : I+MGI was significantly associated with distant recurrence and breast cancer death . The > 50 % of tamoxifen-treated patients categorised as low-risk had <3 % 10-year distant recurrence risk . A continuous risk model ( Breast Cancer Index ( BCI ) ) was developed with the tamoxifen-treated group and the prognostic performance tested in the untreated group was 53 % of patients categorised as low risk with an 8.3 % 10-year distant recurrence risk . Conclusion : Retrospective analysis of this r and omised , prospect i ve trial cohort vali date d the prognostic utility of H : I+MGI and was used to develop and test a continuous risk model that enables prediction of distant recurrence risk at the patient level Clinical biomarker tests that aid in making treatment decisions will play an important role in achieving personalized medicine for cancer patients . Definitive evaluation of the clinical utility of these biomarkers requires conducting large r and omized clinical trials ( RCTs ) . Efficient RCT design is therefore crucial for timely introduction of these medical advances into clinical practice , and a variety of design s have been proposed for this purpose . To guide design and interpretation of RCTs evaluating biomarkers , we present an in-depth comparison of advantages and disadvantages of the commonly used design s. Key aspects of the discussion include efficiency comparisons and special interim monitoring issues that arise because of the complexity of these RCTs . Important ongoing and completed trials are used as examples . We conclude that , in most setting s , r and omized biomarker-stratified design s ( ie , design s that use the biomarker to guide analysis but not treatment assignment ) should be used to obtain a rigorous assessment of biomarker clinical utility BACKGROUND On average , aromatase inhibitors are better than tamoxifen when used as initial or sequential therapy for postmenopausal women with endocrine-responsive early breast cancer . Because there may be contraindications to their use based on side-effects or cost , we investigated subgroups in which aromatase inhibitors may be more or less important . PATIENTS AND METHODS Breast International Group 1 - 98 trial r and omized 6182 women among four groups comparing letrozole and tamoxifen with sequences of each agent ; 5177 ( 84 % ) had central ly confirmed estrogen receptor ( ER ) positivity . We assessed whether central ly determined ER , progesterone receptor ( PgR ) , human epidermal growth factor receptor 2 , and Ki-67 labeling index , alone or in combination with other prognostic features , predicted the magnitude of letrozole effectiveness compared with either sequence or tamoxifen monotherapy . RESULTS Individually , none of the markers significantly predicted differential treatment effects . Sub population treatment effect pattern plot analysis of a composite measure of prognostic risk revealed three patterns . Estimated 5-year disease-free survival  for letrozole monotherapy , letrozole→tamoxifen , tamoxifen→letrozole , and tamoxifen monotherapy were 96 % , 94 % , 93 % , and 94 % , respectively , for patients at lowest risk ; 90 % , 91 % , 93 % , and 86 % , respectively , for patients at intermediate risk ; and 80 % , 76 % , 74 % , and 69 % , respectively , for patients at highest risk . CONCLUSION A composite measure of risk informs treatment selection better than individual biomarkers and supports the choice of 5 years of letrozole for patients at highest risk for recurrence BACKGROUND Predictive markers of response to chemotherapy are lacking in breast cancer patients . Forkhead Box Protein 3 ( FOXP3 ) is an anti-oncogene whose absence in cancer cells could confer resistance to DNA damaging agent . So we made the hypothesis that FOXP3 expression predicts the response to anthracyclines in breast cancer patients and that adjuvant chemotherapy adding taxanes to anthracyclines confers an overall survival ( OS ) benefit over anthracyclines alone , in patients with FOXP3-negative tumors . PATIENTS AND METHODS Expression of FOXP3 in cancer cells was evaluated by immunohistochemistry in tumor sample s from 1097 patients who participated in the PACS01 r and omized trial that evaluated in adjuvant setting the adjunction of docetaxel ( Taxotere ) to anthracyclines in patients with localized breast cancer . Kaplan-Meier analysis and Cox regression model were used to assess OS according to the presence or absence of FOXP3 expression in tumor cells . RESULTS Four hundred and five tumors were found to express FOXP3 ( 37 % ) . FOXP3 expression in breast cancer cells was associated with better OS ( P = 0.003 ) . Uni- and multivariate survival analyses according to treatment arm revealed that FOXP3 expression in breast cancer cells is independently associated with improved OS in patients treated with anthracycline-based adjuvant chemotherapy , but not in patients treated with sequential anthracycline-taxane . Moreover , in vitro experiments showed that FOXP3 induction in breast cancer cell lines using histone deacetylase inhibitor enhances  anthracyclines efficacy . CONCLUSION FOXP3 expression in tumor cells may be an accurate predictive biomarker of anthracycline efficacy in breast cancer HER2 gene amplification and topoisomerase IIα gene ( TOP2A ) alteration have been associated with increased benefit from anthracycline compared to non-anthracycline containing adjuvant breast cancer chemotherapy in some but not other studies . Chromosome 17 centromere ( CEP17 ) duplication was measured on TMAs from formalin-fixed paraffin-embedded specimens obtained from 639 of 716 premenopausal women with node positive breast cancer who received cyclophosphamide , epirubicin and fluorouracil ( CEF ) or cyclophosphamide , methotrexate and fluorouracil ( CMF )  in the r and omized controlled mammary 5 ( MA.5 ) adjuvant trial . The prognostic impact of CEP17 duplication and its interactions with treatment were studied for relapse-free survival ( RFS ) and overall survival ( OS ) . Overall , CEP17 duplication was not significantly associated with RFS or OS in multivariate analysis . For patients whose tumours had normal CEP17 copy number there were no apparent benefits for CEF compared to CMF for RFS ( HR 0.98 ; 95 % CI 0.68–1.42 ) or OS ( HR 1.10 ; 95 % CI 0.72–1.69 ) . For patients whose tumours had CEP17 duplication , there was significant benefit for CEF compared to CMF for RFS ( HR 0.54 ; CI 0.33–0.89 ) and a trend towards significance for OS ( HR 0.64 ; CI 0.37–1.09 ) . The adjusted P values for interaction between treatment and CEP17 duplication were 0.09 for RFS and 0.13 for OS . This study suggests that CEP17 duplication has a borderline association with clinical responsiveness to anthracycline containing chemotherapy similar to previous results seen with HER2 amplification and TOP2A alteration in MA.5 . An appropriately powered meta- analysis is required to discriminate the predictive value of these three c and i date markers Purpose : Residual risk of relapse remains a substantial concern for patients with hormone receptor – positive breast cancer , with approximately half of all disease recurrences occurring after five years of adjuvant antiestrogen therapy . Experimental Design : The objective of this study was to examine the prognostic performance of an optimized model of Breast Cancer Index ( BCI ) , an algorithmic gene expression – based signature , for prediction of early ( 0–5 years ) and late ( > 5 years ) risk of distant recurrence in patients with estrogen receptor – positive ( ER+ ) , lymph node – negative ( LN− ) tumors . The BCI model was vali date d by retrospective analyses of tumor sample s from tamoxifen-treated patients from a r and omized prospect i ve trial ( Stockholm TAM , n = 317 ) and a multi-institutional cohort ( n = 358 ) . Results : Within the Stockholm TAM cohort , BCI risk groups stratified the majority ( ∼65 % ) of patients as low risk with less than 3 % distant recurrence rate for 0 to 5 years and 5 to 10 years . In the multi-institutional cohort , which had larger tumors , 55 % of patients were classified as BCI low risk with less than 5 % distant recurrence rate for 0 to 5 years and 5 to 10 years . For both cohorts , continuous BCI was the most significant prognostic factor beyond st and ard clinicopathologic factors for 0 to 5 years and more than five years . Conclusions : The prognostic sustainability of BCI to assess early- and late-distant recurrence risk at diagnosis has clinical use for decisions of chemotherapy at diagnosis and for decisions for extended adjuvant endocrine therapy beyond five years . Clin Cancer Res ; 19(15 ) ; 4196–205 . © 2013 AACR The cell-cycle regulating protein p27(Kip1 ) ( p27 ) has dual roles by acting as both a cdk inhibitor and as an assembly factor for different cdk complexes . Loss of p27 has been linked to malignant features in tumours ; however , the exact role of p27 deregulation in breast cancer regarding prognostic and treatment predictive information has not been fully clarified . We have evaluated p27 expression in 328 primary , Stage II breast cancers from premenopausal patients who had been r and omised to either tamoxifen treatment or no adjuvant treatment after surgery .  p27 was associated with the oestrogen receptor and cyclin D1 , and p27 downregulation was associated with high proliferation . There was no association between recurrence-free survival ( RFS ) and p27 ( HR = 0.800 , 95 % CI 0.523 - 1.222 , p = 0.300 ) , indicating that p27 is not a prognostic marker . The predictive value of p27 was analysed by comparing RFS in tamoxifen-treated and untreated patients in subgroups of low and high p27 expression ( HR = 0.747 , 95 % CI 0.335 - 1.664 , p = 0.474 and HR = 0.401 , 95 % CI 0.240 - 0.670 , p < 0.001 , respectively ) . Only patients with p27-high tumours benefited from tamoxifen ( multivariate interaction analysis p = 0.034 ) . Our study suggests that p27 downregulation is associated with tamoxifen resistance in premenopausal breast cancer but is not linked to impaired prognosis Treatment selection markers , sometimes called predictive markers , are factors that help clinicians select therapies that maximize good outcomes and minimize adverse outcomes for patients . Existing statistical methods for evaluating a treatment selection marker include assessing its prognostic value , evaluating treatment effects in patients with a restricted range of marker values , and testing for a statistical interaction between marker value and treatment . These methods are inadequate , because they give misleading measures of performance that do not answer key clinical questions about how the marker might help patients choose treatment , how treatment decisions should be made on the basis of a continuous marker measurement , what effect using the marker to select treatment would have on the population , or what proportion of patients would have treatment changes on the basis of marker measurement . Marker-by-treatment predictiveness curves are proposed as a more useful aid to answering these clinical ly relevant questions , because they illustrate treatment effects as a function of marker value , outcomes when using or not using the marker to select treatment , and the proportion of patients for whom treatment recommendations change after marker measurement . R and omized therapeutic clinical trials , in which entry criteria and treatment regimens are not restricted by the marker , are also proposed as the basis for constructing the curves and evaluating and comparing markers BACKGROUND In breast cancer , immunohistochemical assessment of proliferation using the marker Ki67 has potential use in both research and clinical management . However , lack of consistency across laboratories has limited Ki67 's value . A working group was assembled to devise a strategy to harmonize Ki67 analysis and increase scoring concordance . Toward that goal , we conducted a Ki67 reproducibility study . METHODS Eight laboratories received 100 breast cancer cases arranged into 1-mm core tissue microarrays-one  set stained by the participating laboratory and one set stained by the central laboratory  , both using  antibody MIB-1 . Each laboratory scored Ki67 as percentage of positively stained invasive tumor cells using its own method . Six laboratories repeated scoring of 50 locally stained cases on 3 different days . Sources of variation were analyzed using r and om effects models with log2-transformed measurements . Reproducibility was quantified by intraclass correlation coefficient ( ICC ) , and the approximate two-sided 95 % confidence intervals ( CIs ) for the true intraclass correlation coefficients in these experiments were provided . RESULTS Intralaboratory reproducibility was high ( ICC = 0.94 ; 95 % CI = 0.93 to 0.97 ) . Interlaboratory reproducibility was only moderate ( central staining : ICC = 0.71 , 95 % CI = 0.47 to 0.78 ; local staining : ICC = 0.59 , 95 % CI = 0.37 to 0.68 ) . Geometric mean of Ki67 values for each laboratory across the 100 cases ranged 7.1 % to 23.9 % with central staining and 6.1 % to 30.1 % with local staining . Factors contributing to interlaboratory discordance included tumor region selection , counting method , and subjective assessment of staining positivity . Formal counting methods gave more consistent results than visual estimation . CONCLUSIONS Substantial variability in Ki67 scoring was observed among some of the world 's most experienced laboratories . Ki67 values and cutoffs for clinical decision-making can not be transferred between laboratories without st and ardizing scoring methodology because analytical validity is limited PURPOSE Previous pre clinical and clinical data suggest that the immune system influences prognosis and response to chemotherapy ( CT ) ; however , clinical relevance has yet to be established in breast cancer ( BC ) . We hypothesized that increased lymphocytic infiltration would be associated with good prognosis and benefit from immunogenic CT-in this case , anthracycline-only CT-in  selected BC subtypes . PATIENTS AND METHODS We investigated the relationship between quantity and location of lymphocytic infiltrate at diagnosis with clinical outcome in 2009 node-positive BC sample s from the BIG 02 - 98 adjuvant phase III trial comparing anthracycline-only CT ( doxorubicin followed by cyclophosphamide , methotrexate , and fluorouracil [ CMF ] or doxorubicin plus cyclophosphamide followed by CMF ) versus CT combining doxorubicin and docetaxel ( doxorubicin plus docetaxel followed by CMF or doxorubicin followed by docetaxel followed by CMF ) . Readings were independently performed by two pathologists . Disease-free survival ( DFS ) , overall survival ( OS ) , and interaction with type of CT associations were studied . Median follow-up was 8 years . RESULTS There was no significant prognostic association in the global nor estrogen receptor ( ER ) -positive/human epidermal growth factor receptor 2 ( HER2 ) -negative population . However , each 10 % increase in intratumoral and stromal lymphocytic infiltrations was associated with 17 % and 15 % reduced risk of relapse ( adjusted P = .1 and P = .025 ) , respectively , and 27 % and 17 % reduced risk of death in ER-negative/HER2-negative BC regardless of CT type ( adjusted P = .035 and P = .023 ) , respectively . In HER2-positive BC , there was a significant interaction between increasing stromal lymphocytic infiltration ( 10 % increments ) and benefit with anthracycline-only CT ( DFS , interaction P = .042 ; OS , P = .018 ) . CONCLUSION In node-positive , ER-negative/HER2-negative BC , increasing lymphocytic infiltration was associated with excellent prognosis . Further validation of the clinical utility of tumor-infiltrating lymphocytes in this context is warranted . Our data also support the evaluation of immunotherapeutic approaches in selected BC subtypes PURPOSE The 21-gene recurrence score ( RS ) assay quantifies the likelihood of distant recurrence in women with estrogen receptor-positive , lymph node-negative breast cancer treated with adjuvant tamoxifen .  The relationship between the RS and chemotherapy benefit is not known . METHODS The RS was measured in tumors from the tamoxifen-treated and tamoxifen plus chemotherapy-treated patients in the National Surgical Adjuvant Breast and Bowel Project ( NSABP ) B20 trial . Cox proportional hazards models were utilized to test for interaction between chemotherapy treatment and the RS . RESULTS A total of 651 patients were assessable ( 227 r and omly assigned to tamoxifen and 424 r and omly assigned to tamoxifen plus chemotherapy ) . The test for interaction between chemotherapy treatment and RS was statistically significant ( P = .038 ) . Patients with high-RS ( > or = 31 ) tumors ( ie , high risk of recurrence ) had a large benefit from  chemotherapy  ( relative risk , 0.26 ; 95 % CI , 0.13 to 0.53 ; absolute decrease in 10-year distant recurrence rate : mean , 27.6 % ; SE , 8.0 % ) . Patients with low-RS ( < 18 ) tumors derived minimal , if any , benefit from chemotherapy treatment ( relative risk , 1.31 ; 95 % CI , 0.46 to 3.78 ; absolute decrease in distant recurrence rate at 10 years : mean , -1.1 % ; SE , 2.2 % ) . Patients with intermediate-RS tumors did not appear to have a large benefit , but the uncertainty in the estimate can not exclude a clinical ly important benefit . CONCLUSION The RS assay not only quantifies the likelihood of breast cancer recurrence in women with node-negative , estrogen receptor-positive breast cancer , but also predicts the magnitude of  chemotherapy benefit PURPOSE To evaluate the prognostic and predictive value of Ki-67 labeling index ( LI ) in a trial comparing letrozole ( Let ) with tamoxifen ( Tam ) as adjuvant therapy in postmenopausal women with early breast cancer . PATIENTS AND METHODS Breast International Group ( BIG ) trial 1 - 98 r and omly assigned 8,010 patients to four treatment arms comparing Let and Tam with sequences of each agent .  Of 4,922 patients r and omly assigned to receive 5 years of monotherapy with either agent , 2,685 had primary tumor material available for central pathology assessment of Ki-67 LI by immunohistochemistry and had tumors confirmed to express estrogen receptors after central review . The prognostic and predictive value of central ly measured Ki-67 LI on disease-free survival ( DFS ) were assessed among these patients using proportional hazards modeling , with Ki-67 LI values dichotomized at the median value of 11 % . RESULTS Higher values of Ki-67 LI were associated with adverse prognostic factors and with worse DFS ( hazard ratio [ HR ; high : low ] = 1.8 ; 95 % CI , 1.4 to 2.3 ) . The magnitude of the treatment benefit for Let versus Tam was greater among patients with high tumor Ki-67 LI ( HR [ Let : Tam ] = 0.53 ; 95 % CI , 0.39 to 0.72 ) than among patients with low tumor Ki-67 LI ( HR [ Let : Tam ] = 0.81 ; 95 % CI , 0.57 to 1.15 ; interaction P = .09 ) . CONCLUSION Ki-67 LI is confirmed as a prognostic factor in this study . High Ki-67 LI levels may identify a patient group that particularly benefits from initial Let adjuvant therapy BACKGROUND Adjuvant tamoxifen therapy substantially decreases the risk of recurrence and mortality in women with hormone ( estrogen and /or progesterone ) receptor-positive breast cancer . Previous studies have suggested that metabolic conversion of tamoxifen to endoxifen by cytochrome P450 2D6 ( CYP2D6 ) is required for patient benefit from tamoxifen therapy . METHODS Tumor specimens from a subset of postmenopausal patients with hormone receptor-positive early-stage ( stages I , II , and IIIA ) breast cancer , who were enrolled in the r and omized double-blind Arimidex , Tamoxifen , Alone or in Combination ( ATAC ) clinical trial , were genotyped for variants in CYP2D6 ( N = 1203 patients :  anastrozole [ trade name : Arimidex ] group , n = 615 patients ; tamoxifen group , n = 588 patients ) and UDP-glucuronosyltransferase-2B7 ( UGT2B7 ) , whose gene product inactivates endoxifen  ( N = 1209 patients ; anastrozole group , n = 606 patients ;  tamoxifen  group , n = 603 patients ) . Genotyping was performed using polymerase chain reaction-based TaqMan assays . Based on the genotypes for CYP2D6 , patients were classified as poor metabolizer ( PM ) , intermediate metabolizer ( IM ) , or extensive metabolizer ( EM ) phenotypes . We evaluated the association of CYP2D6 and UGT2B7 genotype with distant recurrence ( primary endpoint ) and any recurrence ( secondary endpoint ) by estimating the hazard ratios ( HRs ) and corresponding 95 % confidence intervals ( CIs ) using Cox proportional hazards models . All statistical tests were two-sided . RESULTS After a median follow-up of 10 years , no statistically significant associations were observed between CYP2D6 genotype and recurrence in tamoxifen-treated patients ( PM vs EM : HR for distant recurrence = 1.25 , 95 % CI = 0.55 to 3.15 , P = .64 ; HR for any recurrence = 0.99 , 95 % CI = 0.48 to 2.08 , P = .99 ) . A near-null association was observed between UGT2B7 genotype and recurrence in tamoxifen-treated patients . No associations were observed between CYP2D6 and UGT2B7 genotypes and recurrence in anastrozole-treated patients . CONCLUSION The results do not support the hypothesis that CYP2D6 genotype predicts clinical benefit of adjuvant tamoxifen treatment among postmenopausal breast cancer patients Background . Previous analyses of TOP2A and HER2 in the Danish Breast Cancer Coopererative Group ( DBCG ) trial 89D suggested that TOP2A amplifications and possible also deletions are predictive markers for the effect of adjuvant epirubicin in patients with primary breast cancer . We present an up date d and extended statistical analysis , requested for IVD-labeling of TOP2A testing . Material and methods . In the DBCG trial 89D 980 Danish patients were r and omly assigned to nine cycles of intravenous CMF ( cyclophosphamide , methotrexate , and fluorouracil ) or CEF ( cyclophosphamide , epirubicin , and fluorouracil ) . Archival tumor tissue was collected retrospectively from 806 of these patients in a prospect ively design ed , biological sub- study , and was successfully analyzed for TOP2A aberrations and HER2 status in 773 sample s ( 96 % ) . Recurrence-free survival ( RFS ) was the primary endpoint . Results . TOP2A aberrations ( amplifications and deletions ) were significantly associated with shorter RFS ( p<0.0001 ) and overall survival ( OS ) ( p<0.0001 ) . Deleted cases had worse prognosis than amplified cases . In a Cox proportional hazard model TOP2A was an independent prognostic marker for RFS and OS . Patients with amplifications had a 61 % reduction in the risk of an event ( p=0.002 ) and a 51 % reduction in the risk of death ( p=0.01 ) if allocated to CEF compared to 6 % and 10 % in TOP2A normal patients . A similar but non-significant trend ( p=0.08 ) was shown in patients with TOP2A deletions . Clear statistical evidence of a differential benefit , favoring CEF among patients with TOP2A aberrations was found for RFS ( p=0.02 for interaction ) but not for OS ( p=0.14 for interaction ) . Conclusion . In conclusion , this up date d analysis of TOP2A aberrations in DBCG trial 89D suggests a differential benefit of adjuvant chemotherapy in patients with primary breast cancer , favoring treatment with epirubicin in patients with TOP2A amplifications , and perhaps deletions . Additional studies are needed to clarify the exact importance of TOP2A deletions on outcome , but deletions have proven to be associated with a very poor prognosis BACKGROUND Biomarkers to improve the risk-benefit of extended adjuvant endocrine therapy for late recurrence in patients with oestrogen-receptor-positive breast cancer would be clinical ly valuable . We compared the prognostic ability of the  breast-cancer index  ( BCI ) assay , 21-gene recurrence score ( Oncotype DX ) , and an immunohistochemical prognostic model ( IHC4 ) for both early and late recurrence in patients with oestrogen-receptor-positive , node-negative ( N0 ) disease who took part in the Arimidex , Tamoxifen , Alone or in Combination ( ATAC ) clinical trial . METHODS In this prospect i ve comparison study , we obtained archival tumour blocks from the TransATAC tissue bank from all postmenopausal patients with oestrogen-receptor-positive breast cancer from whom the 21-gene recurrence score and IHC4 values had already been derived . We did BCI analysis in matched sample s with sufficient residual RNA using two BCI models-cubic ( BCI-C ) and linear (BCI-L)-using previously vali date d cutoffs . We assessed prognostic ability of BCI for distant recurrence over 10 years ( the primary endpoint ) and compared it with that of the 21-gene recurrence score and IHC4 . We also tested the ability of the assays to predict early ( 0 - 5 years ) and late ( 5 - 10 years ) distant recurrence . To assess the ability of the biomarkers to predict recurrence beyond st and ard clinicopathological variables , we calculated the change in the likelihood-ratio χ(2 ) ( LR-Δχ(2 ) ) from Cox proportional hazards models . FINDINGS Suitable tissue was available from 665 patients with oestrogen-receptor-positive , N0 breast cancer for BCI analysis . The primary analysis showed significant differences in risk of distant recurrence over 10 years in the categorical BCI-C risk groups ( p<0·0001 ) with 6·8 % ( 95 % CI 4·4 - 10·0 ) of patients in the low-risk group , 17·3 % ( 12·0 - 24·7 ) in the intermediate group , and 22·2 % ( 15·3 - 31·5 ) in the high-risk group having distant recurrence . The secondary analysis showed that BCI-L was a much stronger predictor for overall ( 0 - 10 year ) distant recurrence compared with BCI-C ( interquartile HR 2·30 [ 95 % CI 1·62 - 3·27 ] ; LR-Δχ(2)=22·69 ; p<0·0001 ) . When compared with BCI-L , the 21-gene recurrence score was less predictive ( HR 1·48 [ 95 % CI 1·22 - 1·78 ] ; LR-Δχ(2)=13·68 ; p=0·0002 ) and IHC4 was similar ( HR 1·69 [ 95 % CI 1·51 - 2·56 ] ; LR-Δχ(2)=22·83 ; p<0·0001 ) . All further analyses were done with the BCI-L model . In a multivariable analysis , all assays had significant prognostic ability for early distant recurrence ( BCI-L HR 2·77 [ 95 % CI 1·63 - 4·70 ] , LR-Δχ(2)=15·42 , p<0·0001 ; 21-gene recurrence score HR 1·80 [ 1·42 - 2·29 ] , LR-Δχ(2)=18·48 , p<0·0001 ; IHC4 HR 2·90 [ 2·01 - 4·18 ] , LR-Δχ(2)=29·14 , p<0·0001 ) ; however , only BCI-L was significant for late distant recurrence ( BCI-L HR 1·95 [ 95 % CI 1·22 - 3·14 ] , LR-Δχ(2)=7·97 , p=0·0048 ; 21-gene recurrence score HR 1·13 [ 0·82 - 1·56 ] , LR-Δχ(2)=0·48 , p=0·47 ; IHC4 HR 1·30 [ 0·88 - 1·94 ] , LR-Δχ(2)=1·59 , p=0·20 ) . INTERPRETATION BCI-L was the only significant prognostic test for risk of both early and late distant recurrence and identified two risk population s for each timeframe . It could help to identify patients at high risk for late distant recurrence who might benefit from extended endocrine or other therapy . FUNDING Avon Foundation , National Institutes of Health , Breast Cancer Foundation , US Department of Defense Breast Cancer Research Program , Susan G Komen for the Cure , Breakthrough Breast Cancer through the Mary-Jean Mitchell Green Foundation , AstraZeneca , Cancer Research UK , and the National Institute for Health Research Biomedical Research Centre at the Royal Marsden ( London , UK ) PURPOSE It has been suggested that PTEN , a negative regulator of PI3K/AKT signaling , is involved in tumor sensitivity to trastuzumab . We investigated the association between tumor PTEN protein expression and disease-free survival ( DFS ) of patients r and omly assigned to receive chemotherapy alone ( arm A ) or chemotherapy with sequential ( arm B ) or concurrent trastuzumab ( arm C ) in the phase III early-stage human epidermal growth factor receptor 2 ( HER2 ) -positive trial-North Central Cancer Treatment Group ( NCCTG ) N9831 . PATIENTS AND METHODS The intensity and percentage of invasive cells with cytoplasmic PTEN staining were determined in tissue microarray sections containing three cores per block ( n = 1,286 ) or in whole tissue sections ( WS ; n = 516 ) by using st and ard immunohistochemistry ( 138G6 monoclonal antibody ) . Tumors were considered positive for PTEN ( PTEN-positive ) if any core or WS had any invasive cells with ≥ 1 + staining . Median follow-up was 6.0 years . RESULTS Of 1,802 patients included in this analysis ( of 3,505 patients registered to N9831 ) , 1,342 ( 74 % ) had PTEN-positive tumors . PTEN positivity was associated with hormone receptor negativity ( χ(2 ) P < .001 ) and nodal positivity ( χ(2 ) P = .04 ) . PTEN did not have an impact on DFS within the various arms . Comparing DFS of arm C to arm A , patients with PTEN-positive and PTEN-negative tumors had hazard ratios ( HRs ) of 0.65 ( P = .003 ) and 0.47 ( P = .005 ) , respectively ( interaction P = .16 ) . For arm B versus arm A , patients with PTEN-positive and PTEN-negative tumors had HRs of 0.70 ( P = .009 ) and 0.85 ( P = .44 ) , respectively ( interaction P = .47 ) . CONCLUSION In contrast to selected pre clinical and limited clinical studies suggesting a decrease in trastuzumab sensitivity in patients with PTEN-negative tumors , our data show benefit of adjuvant trastuzumab for patients with HER2-positive breast cancer , independent of tumor PTEN status
target: RECOMMENDATIONS In addition to estrogen and progesterone receptors and human epidermal growth factor receptor 2 , the panel found sufficient evidence of clinical utility for the biomarker assays Oncotype DX , EndoPredict , PAM50 , Breast Cancer Index , and urokinase plasminogen activator and plasminogen activator inhibitor type 1 in specific subgroups of breast cancer .
  No biomarker except for estrogen receptor , progesterone receptor , and human epidermal growth factor receptor 2 was found to guide choices of specific treatment regimens .
background: PURPOSE To provide recommendations on appropriate use of breast tumor biomarker assay results to guide decisions on adjuvant systemic therapy for women with early-stage invasive breast cancer .